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1.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199760

RESUMO

Polymerase chain reaction (PCR) is the standard in nucleic acid amplification technology for infectious disease pathogen detection and has been the primary diagnostic tool employed during the global COVID-19 pandemic. Various PCR technology adaptations, typically using two-oligonucleotide dye-binding methods or three-oligonucleotide hydrolysis probe systems, enable real-time multiplex target detection or single-base specificity for the identification of single-nucleotide polymorphisms (SNPs). A small number of two-oligonucleotide PCR systems facilitating both multiplex detection and SNP identification have been reported; however, these methods often have limitations in terms of target specificity, production of variable or false-positive results, and the requirement for extensive optimisation or post-amplification analysis. This study introduces 3' Tth endonuclease cleavage PCR (3TEC-PCR), a two-oligonucleotide PCR system incorporating a modified primer/probe and a thermostable cleavage enzyme, Tth endonuclease IV, for real-time multiplex detection and SNP identification. Complete analytical specificity, low limits of detection, single-base specificity, and simultaneous multiple target detection have been demonstrated in this study using 3TEC-PCR to identify bacterial meningitis associated pathogens. This is the first report of a two-oligonucleotide, real-time multiplex PCR technology with single-base specificity using Tth endonuclease IV.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , Alelos , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Haemophilus influenzae/genética , Humanos , Meningites Bacterianas/diagnóstico , Meningites Bacterianas/microbiologia , Neisseria meningitidis/genética , Streptococcus pneumoniae/genética
2.
Methods Mol Biol ; 2314: 59-75, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235648

RESUMO

A vast array of molecular biology tools have been developed to investigate the Mycobacterium tuberculosis genome since the advent of its successful sequencing in 1998. These tools, such as quantitative and end point polymerase chain reaction, chromatin immunoprecipitation, and whole genome sequencing, require genomic DNA extracted from lysed mycobacteria. There are numerous methods described in the literature using mechanical, enzymatic, or chemical means to lyse cells and extract genomic DNA to varying degrees of purity. Here, we describe appropriate methods for genomic DNA isolation from solid or liquid cultures from both M. tuberculosis and nontuberculous mycobacteria.


Assuntos
Técnicas Bacteriológicas , Imunoprecipitação da Cromatina , DNA Bacteriano/isolamento & purificação , Mycobacterium/genética , Cetrimônio/química , Reação em Cadeia da Polimerase , Análise de Sequência de DNA
3.
Nat Commun ; 12(1): 4191, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234149

RESUMO

The vaginal and uterine microbiota play important roles in the health of the female reproductive system. However, the interactions among the microbes in these two niches and their effects on uterine health remain unclear. Here we profile the vaginal and uterine microbial samples of 145 women, and combine with deep mining of public data and animal experiments to characterize the microbial translocation in the female reproductive tract and its role in modulating uterine health. Synchronous variation and increasing convergence of the uterine and vaginal microbiome with advancing age are shown. We also find that transplanting certain strains of vaginal bacteria into the vagina of rats induces or reduces endometritis-like symptoms, and verify the damaging or protective effects of certain vaginal bacteria on endometrium. This study clarifies the interdependent relationship of vaginal bacterial translocation with uterine microecology and endometrial health, which will undoubtedly increase our understanding of female reproductive health.


Assuntos
Translocação Bacteriana , Endometrite/microbiologia , Microbiota , Saúde Reprodutiva , Vagina/microbiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Doença Crônica , Estudos de Coortes , DNA Bacteriano/isolamento & purificação , Modelos Animais de Doenças , Endometrite/epidemiologia , Endometrite/patologia , Endométrio/microbiologia , Endométrio/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Fatores de Proteção , RNA Ribossômico 16S/genética , Ratos , Fatores de Risco , Saúde da Mulher , Adulto Jovem
4.
Ticks Tick Borne Dis ; 12(5): 101746, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34091278

RESUMO

Ticks are one of the main vectors of pathogens for humans and animals worldwide. However, they harbor non-pathogenic microorganisms that are important for their survival, facilitating both their nutrition and immunity. We investigated the bacterial communities associated with two neotropical tick species of human and veterinary potential health importance from Brazil: Amblyomma aureolatum and Ornithodoros brasiliensis. In A. aureolatum (adult ticks collected from wild canids from Southern Brazil), the predominant bacterial phyla were Proteobacteria (98.68%), Tenericutes (0.70%), Bacteroidetes (0.14%), Actinobacteria (0.13%), and Acidobacteria (0.05%). The predominant genera were Francisella (97.01%), Spiroplasma (0.70%), Wolbachia (0.51%), Candidatus Midichloria (0.25%), and Alkanindiges (0.13%). The predominant phyla in O. brasiliensis (adults, fed and unfed nymphs collected at the environment from Southern Brazil) were Proteobacteria (90.27%), Actinobacteria (7.38%), Firmicutes (0.77%), Bacteroidetes (0.44%), and Planctomycetes (0.22%). The predominant bacterial genera were Coxiella (87.71%), Nocardioides (1.73%), Saccharopolyspora (0.54%), Marmoricola (0.42%), and Staphylococcus (0.40%). Considering the genera with potential importance for human and animal health which can be transmitted by ticks, Coxiella sp. was found in all stages of O. brasiliensis, Francisella sp. in all stages of A. aureolatum and in unfed nymphs of O. brasiliensis, and Rickettsia sp. in females of A. aureolatum from Banhado dos Pachecos (BP) in Viamão municipality, Brazil, and in females and unfed nymphs of O. brasiliensis. These results deepen our understanding of the tick-microbiota relationship in Ixodidae and Argasidae, driving new studies with the focus on the manipulation of tick microbiota to prevent outbreaks of tick-borne diseases in South America.


Assuntos
Amblyomma/microbiologia , Microbiota , Ornithodoros/microbiologia , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Coxiella/genética , Coxiella/isolamento & purificação , DNA Bacteriano/isolamento & purificação , Francisella/genética , Francisella/isolamento & purificação , Ixodidae/microbiologia , Metagenômica , RNA Ribossômico 16S/genética , Rickettsia/genética , Rickettsia/isolamento & purificação
5.
Medicine (Baltimore) ; 100(25): e26403, 2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34160425

RESUMO

INTRODUCTION: Sepsis and septic shock are the most severe forms of infection affecting predominantly elderly people, preterm and term neonates, and young infants. Even in high-income countries sepsis causes about 8% of admissions to pediatric intensive care units (PICUs). Early diagnosis, rapid anti-infective treatment, and prompt hemodynamic stabilization are crucial for patient survival. In this context, it is essential to identify the causative pathogen as soon as possible to optimize antimicrobial treatment. To date, culture-based diagnostic procedures (e.g., blood cultures) represent the standard of care. However, they have 2 major problems: on the one hand, in the case of very small sample volumes (and thus usually in children), they are not sufficiently sensitive. On the other hand, with a time-to-result of 2 to 5 days, blood cultures need a relatively long time for the anti-infective therapy to be calculated. To overcome these problems, culture-independent molecular diagnostic procedures such as unbiased sequence analysis of circulating cell-free DNA (cfDNA) from plasma samples of septic patients by next-generation sequencing (NGS) have been tested successfully in adult septic patients. However, these results still need to be transferred to the pediatric setting. METHODS: The Next GeneSiPS-Trial is a prospective, observational, non-interventional, multicenter study used to assess the diagnostic performance of an NGS-based approach for the identification of causative pathogens in (preterm and term) neonates (d1-d28, n = 50), infants (d29 to <1 yr, n = 50), and toddlers (1 yr to <5 yr, n = 50) with suspected or proven severe sepsis or septic shock (according to the pediatric sepsis definition) by the use of the quantitative sepsis indicating quantifier (SIQ) score in comparison to standard of care (culture-based) microbiological diagnostics. Potential changes in anti-infective treatment regimens based on these NGS results will be estimated retrospectively by a panel of 3 independent clinical specialists. DISCUSSION: Neonates, infants, and young children are significantly affected by sepsis. Fast and more sensitive diagnostic approaches are urgently needed. This prospective, observational, non-interventional, multicenter study seeks to evaluate an NGS-based approach in critically ill children suffering from sepsis. TRIAL REGISTRATION: DRKS-ID: DRKS00015705 (registered October 24, 2018). https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00015705.


Assuntos
Bacteriemia/diagnóstico , Bactérias/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Técnicas de Diagnóstico Molecular/métodos , Choque Séptico/diagnóstico , Antibacterianos/uso terapêutico , Bacteriemia/tratamento farmacológico , Bacteriemia/microbiologia , Bactérias/genética , Hemocultura , Pré-Escolar , DNA Bacteriano/isolamento & purificação , Diagnóstico Precoce , Feminino , Humanos , Lactente , Recém-Nascido , Unidades de Terapia Intensiva Pediátrica , Masculino , Estudos Multicêntricos como Assunto , Estudos Observacionais como Assunto , Estudos Prospectivos , Estudos Retrospectivos , Índice de Gravidade de Doença , Choque Séptico/sangue , Choque Séptico/tratamento farmacológico , Choque Séptico/microbiologia
6.
BMC Infect Dis ; 21(1): 598, 2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162342

RESUMO

BACKGROUND: Early diagnosis and treatment of Buruli ulcer is critical in order to avoid the debilitating effects of the disease. In this regard, the development of new diagnostic and point of care tools is encouraged. The loop-mediated isothermal amplification for the detection of Mycobacterium ulcerans represents one of the new tools with a good potential of being developed into a point of care test. There is however the need to standardize the assays, reduce sample preparation times, improve the detection/visualization system and optimize them for high-throughput screening, adaptable to low resourced laboratories. METHODS: In this study, we assessed two DNA extraction protocols (modified Boom and EasyNAT methods), three previously published LAMP primer sets (BURULI, MU 2404 and BU-LAMP), and compared the sensitivity and specificity of LAMP assays on three DNA amplification platforms. RESULTS: Our results show that Buruli ulcer diagnosis using primers targeting IS2404 for the LAMP method is sensitive (73.75-91.49%), depending on the DNA extraction method used. Even though the modified Boom DNA extraction method provided the best results, its instrumentation requirement prevent it from being field applicable. The EasyNAT method on the other hand is simpler and may represent the best method for DNA extraction in less resourced settings. CONCLUSIONS: For further work on the development and use of LAMP tests for Buruli diagnosis, it is recommended that the BURULI sets of primers be used, as these yielded the best results in terms of sensitivity (87.50-91.49%) and specificity (89.23-100%), depending on the DNA extraction methods used.


Assuntos
Úlcera de Buruli/diagnóstico , DNA Bacteriano/isolamento & purificação , Técnicas de Diagnóstico Molecular , Mycobacterium ulcerans/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico , Úlcera de Buruli/microbiologia , Primers do DNA , Elementos de DNA Transponíveis , Humanos , Mycobacterium ulcerans/genética , Sistemas Automatizados de Assistência Junto ao Leito , Sensibilidade e Especificidade
7.
Nat Commun ; 12(1): 3254, 2021 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-34059668

RESUMO

The capability to design microbiomes with predictable functions would enable new technologies for applications in health, agriculture, and bioprocessing. Towards this goal, we develop a model-guided approach to design synthetic human gut microbiomes for production of the health-relevant metabolite butyrate. Our data-driven model quantifies microbial interactions impacting growth and butyrate production separately, providing key insights into ecological mechanisms driving butyrate production. We use our model to explore a vast community design space using a design-test-learn cycle to identify high butyrate-producing communities. Our model can accurately predict community assembly and butyrate production across a wide range of species richness. Guided by the model, we identify constraints on butyrate production by high species richness and key molecular factors driving butyrate production, including hydrogen sulfide, environmental pH, and resource competition. In sum, our model-guided approach provides a flexible and generalizable framework for understanding and accurately predicting community assembly and metabolic functions.


Assuntos
Bactérias/metabolismo , Técnicas Bacteriológicas/métodos , Butiratos/metabolismo , Microbioma Gastrointestinal/fisiologia , Anaerobiose , Bactérias/genética , Bactérias/isolamento & purificação , Biologia Computacional , DNA Bacteriano/isolamento & purificação , Genoma Bacteriano , Humanos , Sulfeto de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Microbiologia Industrial/métodos , Engenharia Metabólica , Análise de Sequência de DNA
8.
Nat Commun ; 12(1): 3381, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099669

RESUMO

Nutrient amendment diminished bacterial functional diversity, consolidating carbon flow through fewer bacterial taxa. Here, we show strong differences in the bacterial taxa responsible for respiration from four ecosystems, indicating the potential for taxon-specific control over soil carbon cycling. Trends in functional diversity, defined as the richness of bacteria contributing to carbon flux and their equitability of carbon use, paralleled trends in taxonomic diversity although functional diversity was lower overall. Among genera common to all ecosystems, Bradyrhizobium, the Acidobacteria genus RB41, and Streptomyces together composed 45-57% of carbon flow through bacterial productivity and respiration. Bacteria that utilized the most carbon amendment (glucose) were also those that utilized the most native soil carbon, suggesting that the behavior of key soil taxa may influence carbon balance. Mapping carbon flow through different microbial taxa as demonstrated here is crucial in developing taxon-sensitive soil carbon models that may reduce the uncertainty in climate change projections.


Assuntos
Ciclo do Carbono , Mudança Climática , Nutrientes/metabolismo , Microbiologia do Solo , Solo/química , Acidobacteria/genética , Acidobacteria/isolamento & purificação , Acidobacteria/metabolismo , Biodiversidade , Bradyrhizobium/genética , Bradyrhizobium/isolamento & purificação , Bradyrhizobium/metabolismo , Carbono/metabolismo , DNA Bacteriano/isolamento & purificação , Monitorização de Parâmetros Ecológicos/métodos , Previsões/métodos , Fósforo/metabolismo , RNA Ribossômico 16S/genética , Streptomyces/genética , Streptomyces/isolamento & purificação , Streptomyces/metabolismo
9.
Methods Mol Biol ; 2290: 187-201, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34009591

RESUMO

Polymerase chain reaction (PCR) is a popular molecular tool for detection of bacteria. PCR allows millions of copies of a target segment of DNA to be produced. The DNA is extracted from overnight grown cultures of pure bacterial isolates using either the organo-solvent method or a commercial DNA extraction kit. The quality and purity of the DNA is determined by performing gel electrophoresis on 0.8% agarose gel. The DNA is amplified by performing PCR assay. Bands of approximately 1.5 kb in size are obtained from the amplified products of DNA. The PCR products run on 1.5% agarose gel are visualized with UV light and imaged by gel documentation system. This chapter outlines the protocol for isolation and amplification of DNA from cellulolytic bacteria. Cellulolytic bacteria are considered a potential source of cellulases for pretreatment of crop residues during biogas production. PCR is considered a very powerful, sensitive, specific, fast, and reliable tool in molecular detection and diagnostics.


Assuntos
Biocombustíveis/microbiologia , DNA Bacteriano/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Bacillus/genética , Bactérias/classificação , Bactérias/genética , Cellulomonas/genética , Clostridium/genética , DNA Bacteriano/genética , Eletroforese/métodos , Pseudomonas/genética , Rhodothermus/genética
10.
Medicine (Baltimore) ; 100(21): e26091, 2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34032746

RESUMO

INTRODUCTION: This work reports a patient with recurrent renal calculi subjected to three surgeries in half a year to be in the same position, and the high-throughput sequencing data showed different species in the renal pus and urine samples, which suggested that partial renal infection or stone formation can be judged by the bacteria in urine. PATIENT CONCERNS: The female patient aged 43 years was referred to the authors' department on April 13, 2020, due to left waist pain and fever for 3 days. DIAGNOSIS: Kidney stones and hydronephrosis were determined by a urinary system computed tomography scan. INTERVENTIONS: On April 20, 2020 and June 15, 2020, the patient was successfully treated with left percutaneous nephrolithotomy twice under general anesthesia. An investigation on the health and eating habits of the patient within 6 months was completed at the last admission. The components of the second renal calculus sample were analyzed with an infrared spectrum analyzer. The third renal stone (renal pus, triplicates) was subjected to microbial metagenome sequencing, and urine samples before and after surgery were subjected to 16S RNA sequencing by SEQHEALTH (Wuhan, China). OUTCOMES: After percutaneous nephrolithotomy, the left kidney stones were basically cleared, stone analysis revealed that the main components were calcium oxalate monohydrate, silica, and a small amount of calcium oxalate dehydrate. Although the urine samples exhibited differences, the renal pus and urine sample shared a single species. CONCLUSION: It is not clear that the prospects of partial renal infection or stone formation can be judged by the bacteria in urine.


Assuntos
Infecções por Helicobacter/diagnóstico , Hidronefrose/diagnóstico , Cálculos Renais/diagnóstico , Nefrolitotomia Percutânea/efeitos adversos , Infecções Urinárias/diagnóstico , Adulto , DNA Bacteriano/isolamento & purificação , Feminino , Infecções por Helicobacter/complicações , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/cirurgia , Helicobacter pylori , Humanos , Hidronefrose/microbiologia , Hidronefrose/cirurgia , Cálculos Renais/microbiologia , Cálculos Renais/cirurgia , Metagenoma/genética , RNA Ribossômico 16S/genética , Recidiva , Reoperação , Infecções Urinárias/complicações , Infecções Urinárias/microbiologia , Infecções Urinárias/cirurgia
11.
BMC Infect Dis ; 21(1): 463, 2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34020607

RESUMO

BACKGROUND: Streptococcus pyogenes causes a profound global burden of morbidity and mortality across its diverse clinical spectrum. To support a new controlled human infection ('challenge') model seeking to accelerate S. pyogenes vaccine development, we aimed to develop an accurate and reliable molecular method for quantifying bacterial load from pharyngeal swabs collected during experimental human pharyngitis. METHODS: Combined sequential RNA + DNA extraction from throat swabs was compared to traditional separate RNA-only and DNA-only extractions. An emm-type specific qPCR was developed to detect the emm75 challenge strain. Results from the qPCR were compared to culture, using throat swab samples collected in a human challenge study. RESULTS: The qPCR was 100% specific for the emm75 challenge strain when tested against a panel of S. pyogenes emm-types and other respiratory pathogens. Combined RNA + DNA extraction had similar yield to traditional separate extractions. The combined extraction method and emm75 qPCR had 98.8% sensitivity compared to culture for throat swabs collected from challenge study participants. CONCLUSIONS: We have developed a reliable molecular method for measuring S. pyogenes bacterial load from throat swabs collected in a controlled human infection model of S. pyogenes pharyngitis. TRIAL REGISTRATION: NCT03361163 on 4th December 2017.


Assuntos
Antígenos de Bactérias/genética , Carga Bacteriana , Proteínas da Membrana Bacteriana Externa/genética , Faringite/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Adulto , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Seguimentos , Voluntários Saudáveis , Humanos , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Sensibilidade e Especificidade , Streptococcus pyogenes/classificação , Streptococcus pyogenes/isolamento & purificação
12.
Vet Rec ; 188(9): e143, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33960475

RESUMO

BACKGROUND: Bovine leptospirosis is mainly characterized by reproductive disorders. Contamination of the oocyte was previously demonstrated in vitro, resulting in some apparent damage. However, it is not clear whether it occurs under natural conditions. The present study aimed to characterize the presence of pathogenic Leptospira DNA in the ovarian follicles of non-pregnant cows. METHODS: Follicular fluid samples were collected from 65 animals and subjected to lipL32 PCR and secY sequencing. RESULTS: In total, seven of 65 (10.8%) were positive, indicating a possible early infection of the oocyte. Moreover, secY sequencing identified L. interrogans and L. santarosai, both very closely related to bovine strains from the Sejroe serogroup (Hardjoprjitno and Guaricura). We demonstrated that ovarian follicles can also be infected. CONCLUSIONS: It was hypothesised that ovarian infection can contribute to embryonic death, causing reproductive failure and estrus repetition. In the present study, we show that the organism identified in the follicle is closely related to one that is known to be associated with reproductive disorders.


Assuntos
Doenças dos Bovinos/microbiologia , DNA Bacteriano/isolamento & purificação , Líquido Folicular/microbiologia , Leptospira/genética , Leptospirose/veterinária , Animais , Bovinos , Feminino , Leptospirose/microbiologia , Reação em Cadeia da Polimerase/veterinária , Sorogrupo
13.
Res Vet Sci ; 137: 144-149, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33975193

RESUMO

Feline Vector-Borne Diseases show increased global prevalence and some Anaplasma and Ehrlichia species may pose a risk to human health. The diagnosis of Anaplasma and Ehrlichia species infection in cats is achieved by the combined use of different methods as cytologic examination evidencing intracytoplasmic morulae, serologic tests and molecular assays. The peripheral whole blood is considered the sample of choice for Anaplasma and Ehrlichia species DNA detection in cats, but false negative results are reported leading to underestimation of infection prevalence. In order to have a more accurate assessment of the spread of feline vector-borne pathogens, the presence of Anaplasma spp. and Ehrlichia spp. DNA in 37 owner and shelter cats subjected to necropsy were prospectively investigated by testing in end-point PCR spleen, bone marrow, blood clot and hair samples. The bacteria identified were genetically characterised. Three shelter cats tested positive for A. phagocytophilum DNA in spleen (one cat) or in hair samples (two cats). None of the cats tested positive in bone marrow and blood samples. From the results obtained, it can be assumed that the use of spleen or hair samples could allow a more reliable detection of A. phagocytophilum DNA in cats with blood tested negative. In the phylogeny constructed with a fragment of the heat shock (groEL) gene nucleotide sequences, all the identified A. phagocytophilum clustered with bacteria infecting a wide range of hosts, including humans, showing a potential zoonotic role.


Assuntos
Anaplasma phagocytophilum/isolamento & purificação , Doenças do Gato/microbiologia , Ehrlichiose/veterinária , Cabelo/microbiologia , Baço/microbiologia , Doenças Transmitidas por Vetores/veterinária , Anaplasma phagocytophilum/imunologia , Animais , Anticorpos Antibacterianos/sangue , Gatos , DNA Bacteriano/isolamento & purificação , Feminino , Masculino , Técnicas de Diagnóstico Molecular/veterinária , Doenças Transmitidas por Vetores/diagnóstico , Doenças Transmitidas por Vetores/microbiologia
14.
Nat Commun ; 12(1): 3186, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-34045458

RESUMO

Long-term infection of the airways of cystic fibrosis patients with Pseudomonas aeruginosa is often accompanied by a reduction in bacterial growth rate. This reduction has been hypothesised to increase within-patient fitness and overall persistence of the pathogen. Here, we apply adaptive laboratory evolution to revert the slow growth phenotype of P. aeruginosa clinical strains back to a high growth rate. We identify several evolutionary trajectories and mechanisms leading to fast growth caused by transcriptional and mutational changes, which depend on the stage of adaptation of the strain. Return to high growth rate increases antibiotic susceptibility, which is only partially dependent on reversion of mutations or changes in the transcriptional profile of genes known to be linked to antibiotic resistance. We propose that similar mechanisms and evolutionary trajectories, in reverse direction, may be involved in pathogen adaptation and the establishment of chronic infections in the antibiotic-treated airways of cystic fibrosis patients.


Assuntos
Antibacterianos/farmacologia , Fibrose Cística/complicações , Resistência Microbiana a Medicamentos/genética , Evolução Molecular , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/genética , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/genética , Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Fibrose Cística/tratamento farmacológico , Fibrose Cística/imunologia , Fibrose Cística/microbiologia , Análise Mutacional de DNA , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Evolução Molecular Direcionada , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica , Aptidão Genética/efeitos dos fármacos , Genoma Bacteriano , Humanos , Pulmão/imunologia , Pulmão/microbiologia , Testes de Sensibilidade Microbiana , Mutação , Fenótipo , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação , Escarro/microbiologia
15.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946648

RESUMO

Growing evidence highlights the crucial role of gut microbiota in affecting different aspects of obesity. Considering the ability of deep transcranial magnetic stimulation (dTMS) to modulate the cortical excitability, the reward system, and, indirectly, the autonomic nervous system (ANS), we hypothesized a potential role of dTMS in affecting the brain-gut communication pathways, and the gut microbiota composition in obesity. In a hospital setting, 22 subjects with obesity (5 M, 17 F; 44.9 ± 2.2 years; BMI 37.5 ± 1.0 kg/m2) were randomized into three groups receiving 15 sessions (3 per week for 5 weeks) of high frequency (HF), low frequency (LF) dTMS, or sham stimulation. Fecal samples were collected at baseline and after 5 weeks of treatment. Total bacterial DNA was extracted from fecal samples using the QIAamp DNA Stool Mini Kit (Qiagen, Italy) and analyzed by a metagenomics approach (Ion Torrent Personal Genome Machine). After 5 weeks, a significant weight loss was found in HF (HF: -4.1 ± 0.8%, LF: -1.9 ± 0.8%, sham: -1.3 ± 0.6%, p = 0.042) compared to LF and sham groups, associated with a decrease in norepinephrine compared to baseline (HF: -61.5 ± 15.2%, p < 0.01; LF: -31.8 ± 17.1%, p < 0.05; sham: -35.8 ± 21.0%, p > 0.05). Furthermore, an increase in Faecalibacterium (+154.3% vs. baseline, p < 0.05) and Alistipes (+153.4% vs. baseline, p < 0.05) genera, and a significant decrease in Lactobacillus (-77.1% vs. baseline, p < 0.05) were found in HF. Faecalibacterium variations were not significant compared to baseline in the other two groups (LF: +106.6%, sham: +27.6%; p > 0.05) as well as Alistipes (LF: -54.9%, sham: -15.1%; p > 0.05) and Lactobacillus (LF: -26.0%, sham: +228.3%; p > 0.05) variations. Norepinephrine change significantly correlated with Bacteroides (r2 = 0.734; p < 0.05), Eubacterium (r2 = 0.734; p < 0.05), and Parasutterella (r2 = 0.618; p < 0.05) abundance variations in HF. In conclusion, HF dTMS treatment revealed to be effective in modulating gut microbiota composition in subjects with obesity, reversing obesity-associated microbiota variations, and promoting bacterial species representative of healthy subjects with anti-inflammatory properties.


Assuntos
Microbioma Gastrointestinal , Obesidade/microbiologia , Obesidade/terapia , Estimulação Magnética Transcraniana/métodos , Adulto , Idoso , Sistema Nervoso Autônomo/fisiopatologia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Biodiversidade , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Método Duplo-Cego , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Norepinefrina/sangue , Obesidade/fisiopatologia , RNA Ribossômico 16S/genética , Fatores de Tempo , Perda de Peso , Adulto Jovem
16.
J Dermatol Sci ; 102(2): 109-115, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33893030

RESUMO

BACKGROUND: The gut microbiota is known to play a key role in autoimmune diseases. OBJECTIVES: To identify and compare the characteristics in the gut microbial composition of patients with alopecia areata (AA) and healthy controls (HCs). METHODS: In a cross-sectional discovery cohort, we enrolled 33 patients with AA and 35 HCs from the same geographic location in Shanghai, China. The 16S rRNA gene sequencing and bioinformatic analyses were conducted to analyze DNA extracted from the subjects. RESULTS: The α-diversity of the AA group demonstrated no statistically significant differences compared with the HC group (P > 0.05). However, the overall gut microbial communities in the AA group were distinct from the HCs (P = 0.0096). We also adopted a random forest model to select three AA-associated OTU biomarkers: OTU1237(Achromobacter), OTU257(Megasphaera), and OTU1784(Lachnospiraceae Incertae Sedis). CONCLUSION: The overall gut microbial composition for AA was distinct from that of HCs. The gut microbial markers we identified may potentially be used for earlier diagnosis and as therapeutic targets.


Assuntos
Alopecia em Áreas/diagnóstico , Microbioma Gastrointestinal/imunologia , Adulto , Alopecia em Áreas/imunologia , Alopecia em Áreas/microbiologia , Biomarcadores/análise , China , Estudos Transversais , DNA Bacteriano/isolamento & purificação , Diagnóstico Precoce , Feminino , Microbioma Gastrointestinal/genética , Voluntários Saudáveis , Humanos , Masculino , Filogenia , RNA Ribossômico 16S/genética , Adulto Jovem
17.
Nutrients ; 13(4)2021 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-33924396

RESUMO

BACKGROUND: Few preclinical studies have shown that Knee osteoarthritis (KOA) is linked to gut microbiome dysbiosis and chronic inflammation. This pilot study was designed to look at the gut microbiome composition in KOA patients and normal individuals with or without vitamin D deficiency (VDD, serum vitamin D <30 ng/mL). METHODS: This pilot study was conducted prospectively in 24 participants. The faecal samples of all the participants were taken for DNA extraction. The V3-V4 region of 16s rRNA was amplified, and the library was prepared and sequenced on the Illumina Miseq platform. RESULTS: The mean (±SD) age was 45.5 (±10.2) years with no defined comorbidities. Of 447 total Operational Taxonomic Units (OTUs), a differential abundance of 16 nominally significant OTUs between the groups was observed. Linear discriminate analysis (LEfSe) revealed a significant difference in bacteria among the study groups. Pseudobutyrivibrio and Odoribacter were specific for VDD, while Parabacteroides, Butyricimonas and Gordonibacter were abundant in the KOA_VDD group, and Peptococcus, Intestimonas, Delftia and Oribacterium were abundant in the KOA group. About 80% of bacterial species were common among different groups and hence labelled as core bacterial species. However, the core microbiome of KOA and VDD groups were not seen in the KOA_VDD group, suggesting that these bacterial groups were affected by the interaction of the KOA and VDD factors. CONCLUSION: Parabacteroides, Butyricimonas, Pseudobutyrivibrio, Odoribacter and Gordonibacter are the predominant bacteria in vitamin D deficient patients with or without KOA. Together these results indicate an association between the gut microbiome, vitamin D and knee osteoarthritis.


Assuntos
Disbiose/complicações , Microbioma Gastrointestinal/imunologia , Osteoartrite do Joelho/imunologia , Deficiência de Vitamina D/imunologia , Adulto , DNA Bacteriano/isolamento & purificação , Disbiose/diagnóstico , Disbiose/imunologia , Disbiose/microbiologia , Fezes/microbiologia , Microbioma Gastrointestinal/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/microbiologia , Filogenia , Projetos Piloto , RNA Ribossômico 16S/genética , Vitamina D/sangue , Deficiência de Vitamina D/sangue , Deficiência de Vitamina D/diagnóstico , Deficiência de Vitamina D/microbiologia
18.
Int J Mol Sci ; 22(8)2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923762

RESUMO

The efficiency of existing cell lysis methods to isolate nucleic acids from diverse bacteria varies depending on cell wall structures. This study tested a novel idea of using broad-spectrum antimicrobial peptides to improve the lytic efficiency of hard-to-lyse bacteria and characterized their differences. The lysis conditions of Staphylococcus aureus using recombinant porcine myeloid antimicrobial peptide 36 (PMAP-36), a broad-spectrum pig cathelicidin, was optimized, and RNA isolation was performed with cultured pellets of ten bacterial species using various membranolytic proteins. Additionally, three other antimicrobial peptides, protegrin-1 (PG-1), melittin, and nisin, were evaluated for their suitability as the membranolytic agents of bacteria. However, PMAP-36 use resulted in the most successful outcomes in RNA isolation from diverse bacterial species. The amount of total RNA obtained using PMAP-36 increased by ~2-fold compared to lysozyme in Salmonella typhimurium. Streptococci species were refractory to all lytic proteins tested, although the RNA yield from PMAP-36 treatment was slightly higher than that from other methods. PMAP-36 use produced high-quality RNA, and reverse transcription PCR showed the efficient amplification of the 16S rRNA gene from all tested strains. Additionally, the results of genomic DNA isolation were similar to those of RNA isolation. Thus, our findings present an additional option for high quality and unbiased nucleic acid isolation from microbiomes or challenging bacterial strains.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , RNA Bacteriano/química , Staphylococcus aureus/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Fracionamento Celular/métodos , Fracionamento Celular/normas , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , RNA Bacteriano/isolamento & purificação , Staphylococcus aureus/efeitos dos fármacos
19.
Nat Commun ; 12(1): 2126, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33837203

RESUMO

There is accumulating evidence that the lower airway microbiota impacts lung health. However, the link between microbial community composition and lung homeostasis remains elusive. We combine amplicon sequencing and bacterial culturing to characterize the viable bacterial community in 234 longitudinal bronchoalveolar lavage samples from 64 lung transplant recipients and establish links to viral loads, host gene expression, lung function, and transplant health. We find that the lung microbiota post-transplant can be categorized into four distinct compositional states, 'pneumotypes'. The predominant 'balanced' pneumotype is characterized by a diverse bacterial community with moderate viral loads, and host gene expression profiles suggesting immune tolerance. The other three pneumotypes are characterized by being either microbiota-depleted, or dominated by potential pathogens, and are linked to increased immune activity, lower respiratory function, and increased risks of infection and rejection. Collectively, our findings establish a link between the lung microbial ecosystem, human lung function, and clinical stability post-transplant.


Assuntos
Rejeição de Enxerto/microbiologia , Transplante de Pulmão/efeitos adversos , Pulmão/microbiologia , Microbiota/imunologia , Pneumonia Bacteriana/microbiologia , Adulto , Aloenxertos/imunologia , Aloenxertos/microbiologia , Bactérias/genética , Bactérias/imunologia , Bactérias/isolamento & purificação , Bactérias/patogenicidade , Carga Bacteriana/imunologia , Técnicas Bacteriológicas , Líquido da Lavagem Broncoalveolar/microbiologia , Broncoscopia , DNA Bacteriano/isolamento & purificação , Feminino , Rejeição de Enxerto/diagnóstico , Rejeição de Enxerto/imunologia , Humanos , Tolerância Imunológica , Estudos Longitudinais , Pulmão/imunologia , Masculino , Metagenômica , Microbiota/genética , Pessoa de Meia-Idade , Pneumonia Bacteriana/diagnóstico , Pneumonia Bacteriana/imunologia , Estudos Prospectivos , RNA Ribossômico 16S/genética
20.
Nat Commun ; 12(1): 2267, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33859184

RESUMO

Studies in humans and laboratory animals link stable gut microbiome "enterotypes" with long-term diet and host health. Understanding how this paradigm manifests in wild herbivores could provide a mechanistic explanation of the relationships between microbiome dynamics, changes in dietary resources, and outcomes for host health. We identify two putative enterotypes in the African buffalo gut microbiome. The enterotype prevalent under resource-abundant dietary regimes, regardless of environmental conditions, has high richness, low between- and within-host beta diversity, and enrichment of genus Ruminococcaceae-UCG-005. The second enterotype, prevalent under restricted dietary conditions, has reduced richness, elevated beta diversity, and enrichment of genus Solibacillus. Population-level gamma diversity is maintained during resource restriction by increased beta diversity between individuals, suggesting a mechanism for population-level microbiome resilience. We identify three pathogens associated with microbiome variation depending on host diet, indicating that nutritional background may impact microbiome-pathogen dynamics. Overall, this study reveals diet-driven enterotype plasticity, illustrates ecological processes that maintain microbiome diversity, and identifies potential associations between diet, enterotype, and disease.


Assuntos
Búfalos/microbiologia , Doenças Transmissíveis/veterinária , Comportamento Alimentar/fisiologia , Microbioma Gastrointestinal/imunologia , Animais , Búfalos/fisiologia , Doenças Transmissíveis/epidemiologia , Doenças Transmissíveis/microbiologia , DNA Bacteriano/isolamento & purificação , Fezes/microbiologia , Firmicutes/genética , Firmicutes/isolamento & purificação , Incidência , Metagenômica , Filogenia , Planococáceas/genética , Planococáceas/isolamento & purificação , Prevalência , RNA Ribossômico 16S/genética , África do Sul/epidemiologia , Simbiose/imunologia
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