Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.459
Filtrar
1.
J Med Microbiol ; 70(9)2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34477546

RESUMO

Introduction. Carbapenem-resistant enterobacterales (CRE) are listed among the most urgent antibiotic resistance threats.Hypothesis. Previous studies on the mechanisms of CRE in Kuwait have focused on carbapenemases. There have been no studies on non-carbapenemase-producing CRE in Kuwait.Aim/Gap Statement. The aim of this study was to investigate the genetic characteristics of non-carbapenemase-producing carbapenem-resistant enterobacterales (NCPE) isolates using whole-genome sequencing (WGS).Methodology. Fourteen confirmed NCPE isolates that were negative for genes encoding carbapenemase production by polymerase chain reaction (PCR) assays using rectal swabs from intensive care unit patients were characterized using phenotypic, PCR and WGS methods. Susceptibility testing was performed via Etest and clonality via multi-locus sequence typing (MLST).Results. All of the isolates were resistant to ertapenem; 78.6 % were resistant to imipenem, meropenem and trimethoprim-sulfamethoxazole. Resistance to the other antibiotics was variable, ranging from 28.5 (colistin) through 50 (tigecycline) and 64.3 (amikacin) up to 85.7 % against both amoxicillin-clavulanic acid and ciprofloxacin. WGS detected several resistance genes mediating the production of ß-lactamases, genes encoding an outer-membrane porin permeability mutation resulting in reduced susceptibility to ß-lactams, including carbapenems, and genes for multidrug-resistant (MDR) efflux pumps. The isolates also possessed global activator protein MarA, which mediated reduced permeability to ß-lactams. The existence of ß-lactamase genes, overexpression of MDR efflux pumps and reduced permeability mediated by the porin genes were responsible for carbapenem resistance.Conclusions. This finding reflects the superior detection capabilities offered by WGS analysis, which can be used to complement traditional methods and overcome their limited resolution in clinical settings.


Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos , Farmacorresistência Bacteriana/genética , Infecções por Enterobacteriaceae/microbiologia , Trato Gastrointestinal/microbiologia , Sequenciamento Completo do Genoma/métodos , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Humanos , Kuweit/epidemiologia
2.
Viruses ; 13(7)2021 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-34372544

RESUMO

Porcine deltacoronavirus (PDCoV), a highly transmissible intestinal pathogen, causes mild to severe clinical symptoms, such as anorexia, vomiting and watery diarrhea, in piglets and/or sows. Since the first report of PDCoV infection in Hong Kong in 2012, the virus has readily disseminated to North America and several countries in Asia. However, to date, no unified phylogenetic classification system has been developed. To fill this gap, we classified historical PDCoV reference strains into two major genogroups (G-I and G-II) and three subgroups (G-II-a, G-II-b and G-II-c). In addition, no genetic research on the whole PDCoV genome or spike gene has been conducted on isolates from Taiwan so far. To delineate the genetic characteristics of Taiwanese PDCoV, we performed whole-genome sequencing to decode the viral sequence. The PDCoV/104-553/TW-2015 strain is closely related to the G-II-b group, which is mainly composed of PDCoV variants from China. Additionally, various mutations in the Taiwanese PDCoV (104-553/TW-2015) strain might be linked to the probability of recombination with other genogroups of PDCoVs or other porcine coronaviruses. These results represent a pioneering phylogenetic characterization of the whole genome of a PDCoV strain isolated in Taiwan in 2015 and will potentially facilitate the development of applicable preventive strategies against this problematic virus.


Assuntos
Deltacoronavirus/classificação , Deltacoronavirus/genética , Suínos/virologia , Animais , Coronavirus/genética , Infecções por Coronavirus/virologia , Diarreia/genética , Diarreia/virologia , Fezes/virologia , Filogenia , Doenças dos Suínos/virologia , Taiwan , Sequenciamento Completo do Genoma/métodos
3.
Int J Mol Sci ; 22(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34445389

RESUMO

DyP-type peroxidases are a family of heme peroxidases named for their ability to degrade persistent anthraquinone dyes. DyP-type peroxidases are subclassified into three classes: classes P, I and V. Based on its genome sequence, Streptomyces avermitilis, eubacteria, has two genes presumed to encode class V DyP-type peroxidases and two class I genes. We have previously shown that ectopically expressed SaDyP2, a member of class V, indeed has the characteristics of a DyP-type peroxidase. In this study, we analyzed SaDyP1, a member of the same class V as SaDyP2. SaDyP1 showed high amino acid sequence identity to SaDyP2, retaining a conserved GXXDG motif and catalytic aspartate. SaDyP1 degraded anthraquinone dyes, which are specific substrates of DyP-type peroxidases but not azo dyes. In addition to such substrate specificity, SaDyP1 showed other features of DyP-type peroxidases, such as low optimal pH. Furthermore, immunoblotting using an anti-SaDyP2 polyclonal antibody revealed that SaDyP1 and/or SaDyP2 is expressed in mycelia of wild-type S. avermitilis.


Assuntos
Peroxidases/genética , Peroxidases/metabolismo , Streptomyces/enzimologia , Sequenciamento Completo do Genoma/métodos , Motivos de Aminoácidos , Sequência de Aminoácidos , Antraquinonas/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Genoma Bacteriano , Concentração de Íons de Hidrogênio , Modelos Moleculares , Peroxidases/química , Conformação Proteica , Streptomyces/genética , Termodinâmica
4.
mSphere ; 6(4): e0038921, 2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34346709

RESUMO

SARS-CoV-2 nosocomial outbreaks in the first COVID-19 wave were likely associated with a shortage of personal protective equipment and scarce indications on control measures. Having covered these limitations, updates on current SARS-CoV-2 nosocomial outbreaks are required. We carried out an in-depth analysis of a 27-day nosocomial outbreak in a gastroenterology ward in our hospital, potentially involving 15 patients and 3 health care workers. Patients had stayed in one of three neighboring rooms in the ward. The severity of the infections in six of the cases and a high fatality rate made the clinicians suspect the possible involvement of a single virulent strain persisting in those rooms. Whole-genome sequencing (WGS) of the strains from 12 patients and 1 health care worker revealed an unexpected complexity. Five different SARS-CoV-2 strains were identified, two infecting a single patient each, ruling out their relationship with the outbreak; the remaining three strains were involved in three independent, overlapping, limited transmission clusters with three, three, and five cases. Whole-genome sequencing was key to understand the complexity of this outbreak. IMPORTANCE We report a complex epidemiological scenario of a nosocomial COVID-19 outbreak in the second wave, based on WGS analysis. Initially, standard epidemiological findings led to the assumption of a homogeneous outbreak caused by a single SARS-CoV-2 strain. The discriminatory power of WGS offered a strikingly different perspective consisting of five introductions of different strains, with only half of them causing secondary cases in three independent overlapping clusters. Our study exemplifies how complex the SARS-CoV-2 transmission in the nosocomial setting during the second COVID-19 wave occurred and leads to extending the analysis of outbreaks beyond the initial epidemiological assumptions.


Assuntos
COVID-19/epidemiologia , COVID-19/transmissão , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/transmissão , SARS-CoV-2/patogenicidade , Adolescente , Adulto , Idoso , COVID-19/virologia , Infecção Hospitalar/virologia , Surtos de Doenças/prevenção & controle , Feminino , Genoma Viral/genética , Pessoal de Saúde , Hospitais , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , SARS-CoV-2/genética , Sequenciamento Completo do Genoma/métodos , Adulto Jovem
5.
Methods Mol Biol ; 2327: 119-137, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34410643

RESUMO

Outbreak analysis and transmission surveillance of viruses can be performed via whole-genome sequencing after viral isolation. Such techniques have recently been applied to characterize and monitor SARS-CoV-2 , the etiological agent of the COVID-19 pandemic. However, the isolation and culture of SARS-CoV-2 is time consuming and requires biosafety level 3 containment, which is not ideal for many resource-constrained settings. An alternate method, bait capture allows target enrichment and sequencing of the entire SARS-CoV-2 genome eliminating the need for viral culture. This method uses a set of hybridization probes known as "baits" that span the genome and provide sensitive, accurate, and minimal off-target hybridization. Baits can be designed to detect any known virus or bacteria in a wide variety of specimen types, including oral secretions. The bait capture method presented herein allows the whole genome of SARS-CoV-2 in saliva to be sequenced without the need to culture and provides an outline of bait design and bioinformatic analysis to guide a bioinformatician.


Assuntos
Genoma Viral , SARS-CoV-2/genética , Saliva/virologia , Sequenciamento Completo do Genoma/métodos , Biologia Computacional , DNA Complementar/genética , Humanos , Sondas Moleculares/genética , Reação em Cadeia da Polimerase/métodos , SARS-CoV-2/isolamento & purificação , Manejo de Espécimes/métodos , Estreptavidina , Sequenciamento Completo do Genoma/instrumentação
6.
Trop Biomed ; 38(3): 283-288, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34362871

RESUMO

Various methods have been developed for rapid and high throughput full genome sequencing of SARS-CoV-2. Here, we described a protocol for targeted multiplex full genome sequencing of SARS-CoV-2 genomic RNA directly extracted from human nasopharyngeal swabs using the Ion Personal Genome Machine (PGM). This protocol involves concomitant amplification of 237 gene fragments encompassing the SARS-CoV-2 genome to increase the abundance and yield of viral specific sequencing reads. Five complete and one near-complete genome sequences of SARS-CoV-2 were generated with a single Ion PGM sequencing run. The sequence coverage analysis revealed two amplicons (positions 13 751-13 965 and 23 941-24 106), which consistently gave low sequencing read coverage in all isolates except 4Apr20-64- Hu. We analyzed the potential primer binding sites within these low covered regions and noted that the 4Apr20-64-Hu possess C at positions 13 730 and 23 929, whereas the other isolates possess T at these positions. The genome nucleotide variations observed suggest that the naturally occurring variations present in the actively circulating SARS-CoV-2 strains affected the performance of the target enrichment panel of the Ion AmpliSeq™ SARS CoV 2 Research Panel. The possible impact of other genome nucleotide variations warrants further investigation, and an improved version of the Ion AmpliSeq™ SARS CoV 2 Research Panel, hence, should be considered.


Assuntos
Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Reação em Cadeia da Polimerase Multiplex , SARS-CoV-2/genética , Sequenciamento Completo do Genoma , Sequência de Bases , COVID-19 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos , Sequenciamento Completo do Genoma/métodos
7.
Viruses ; 13(7)2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34372564

RESUMO

Avian bornaviruses were first described in 2008 as the causative agents of proventricular dilatation disease (PDD) in parrots and their relatives (Psittaciformes). To date, 15 genetically highly diverse avian bornaviruses covering at least five viral species have been discovered in different bird orders. Currently, the primary diagnostic tool is the detection of viral RNA by conventional or real-time RT-PCR (rRT-PCR). One of the drawbacks of this is the usage of either specific assays, allowing the detection of one particular virus, or of assays with a broad detection spectrum, which, however, do not allow for the simultaneous specification of the detected virus. To facilitate the simultaneous detection and specification of avian bornaviruses, a multiplex real-time RT-PCR assay was developed. Whole-genome sequences of various bornaviruses were aligned. Primers were designed to recognize conserved regions within the overlapping X/P gene and probes were selected to detect virus species-specific regions within the target region. The optimization of the assay resulted in the sensitive and specific detection of bornaviruses of Psittaciformes, Passeriformes, and aquatic birds. Finally, the new rRT-PCR was successfully employed to detect avian bornaviruses in field samples from various avian species. This assay will serve as powerful tool in epidemiological studies and will improve avian bornavirus detection.


Assuntos
Bornaviridae/genética , Bornaviridae/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Animais , Doenças das Aves/virologia , Aves/genética , Aves/virologia , Primers do DNA/genética , Genoma Viral , Infecções por Mononegavirales/veterinária , Papagaios/genética , Papagaios/virologia , Passeriformes/genética , Passeriformes/virologia , Filogenia , RNA Viral/genética , Sequenciamento Completo do Genoma/métodos
8.
PLoS One ; 16(8): e0255691, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34351998

RESUMO

Accurate and timely diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is clinically essential, and is required also to monitor confirmed cases aiming to prevent further spread. Positive real-time PCR results at late time points following initial diagnosis may be clinically misleading as this methodology cannot account for the infection capabilities and the existence of whole genome sequences. In this study, 47 serial respiratory samples were tested by Allplex-nCoV test (Seegene), a triplex of three assays targeting the SARS-CoV-2 RdRP, E and N genes and subsequently assessed by next generation sequencing (NGS). COVID19 patients were tested at an early stage of the disease, when all these viral gene targets were positive, and at an advanced stage, when only the N gene target was positive in the Allplex-nCoV test. The corresponding NGS results showed the presence of complete viral genome copies at both early and advanced stages of the disease, although the total number of mapped sequences was lower in samples from advanced disease stages. We conclude that reduced viral transmission at this late disease stage may result from the low quantities of complete viral sequences and not solely from transcription favoring the N gene.


Assuntos
COVID-19/genética , SARS-CoV-2/genética , Sequenciamento Completo do Genoma/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , COVID-19/diagnóstico , Feminino , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/patogenicidade
9.
Methods Mol Biol ; 2351: 353-368, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382200

RESUMO

DNA methylation can regulate gene expression by modulating chromatin accessibility and transcription factor binding on promoter and enhancer regions. Whole-genome bisulfite sequencing (WGBS) represents the most informative and comprehensive analysis to profile the DNA methylation status of all the cytosines at single-base resolution. However, most of the available protocols recommend an amount of input DNA (50 ng-5µg) that makes the WGBS unsuitable for limited samples and cell populations. In this chapter, we provide complete protocol to perform WGBS libraries from very low-input DNA. This protocol is recommended for the analysis of the whole-genome DNA methylation pattern in rare cell populations, like a defined stem cell population isolated from animal models or human samples.


Assuntos
Metilação de DNA , Epigênese Genética , Epigenômica/métodos , Sequenciamento Completo do Genoma/métodos , Biologia Computacional/métodos , Ilhas de CpG , Elementos Facilitadores Genéticos , Biblioteca Gênica , Técnicas de Amplificação de Ácido Nucleico , Regiões Promotoras Genéticas , Software
10.
Nat Commun ; 12(1): 4612, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34326338

RESUMO

Metastatic and locally-advanced neuroendocrine neoplasms (aNEN) form clinically and genetically heterogeneous malignancies, characterized by distinct prognoses based upon primary tumor localization, functionality, grade, proliferation index and diverse outcomes to treatment. Here, we report the mutational landscape of 85 whole-genome sequenced aNEN. This landscape reveals distinct genomic subpopulations of aNEN based on primary localization and differentiation grade; we observe relatively high tumor mutational burdens (TMB) in neuroendocrine carcinoma (average 5.45 somatic mutations per megabase) with TP53, KRAS, RB1, CSMD3, APC, CSMD1, LRATD2, TRRAP and MYC as major drivers versus an overall low TMB in neuroendocrine tumors (1.09). Furthermore, we observe distinct drivers which are enriched in somatic aberrations in pancreatic (MEN1, ATRX, DAXX, DMD and CREBBP) and midgut-derived neuroendocrine tumors (CDKN1B). Finally, 49% of aNEN patients reveal potential therapeutic targets based upon actionable (and responsive) somatic aberrations within their genome; potentially directing improvements in aNEN treatment strategies.


Assuntos
Mutação , Tumores Neuroendócrinos/genética , Idoso , Feminino , Genes Neoplásicos , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Tumores Neuroendócrinos/classificação , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/patologia , Prognóstico , Sequenciamento Completo do Genoma/métodos
11.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209998

RESUMO

Acne vulgaris, which is mostly associated with the colonization of Cutibacterium acnes (C. acnes), is a common skin inflammatory disease in teenagers. However, over the past few years, the disease has extended beyond childhood to chronically infect approximately 40% of adults. While antibiotics have been used for several decades to treat acne lesions, antibiotic resistance is a growing crisis; thus, finding a new therapeutic target is urgently needed. Studies have shown that phage therapy may be one alternative for treating multi-drug-resistant bacterial infections. In the present study, we successfully isolated a C. acnes phage named TCUCAP1 from the skin of healthy volunteers. Morphological analysis revealed that TCUCAP1 belongs to the family Siphoviridae with an icosahedral head and a non-contractile tail. Genome analysis found that TCUCAP1 is composed of 29,547 bp with a G+C content of 53.83% and 56 predicted open reading frames (ORFs). The ORFs were associated with phage structure, packing, host lysis, DNA metabolism, and additional functions. Phage treatments applied to mice with multi-drug-resistant (MDR) C.-acnes-induced skin inflammation resulted in a significant decrease in inflammatory lesions. In addition, our attempt to formulate the phage into hydroxyethyl cellulose (HEC) cream may provide new antibacterial preparations for human infections. Our results demonstrate that TCUCAP1 displays several features that make it an ideal candidate for the control of C. acnes infections.


Assuntos
Acne Vulgar/terapia , Terapia por Fagos/métodos , Propionibacterium acnes/virologia , Siphoviridae/classificação , Sequenciamento Completo do Genoma/métodos , Acne Vulgar/microbiologia , Animais , Composição de Bases , Celulose/química , Modelos Animais de Doenças , Composição de Medicamentos , Farmacorresistência Bacteriana Múltipla , Tamanho do Genoma , Genoma Viral , Voluntários Saudáveis , Humanos , Injeções Intradérmicas , Camundongos , Fases de Leitura Aberta , Filogenia , Propionibacterium acnes/fisiologia , Siphoviridae/genética , Siphoviridae/isolamento & purificação , Pele/virologia
12.
Genome Med ; 13(1): 121, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34321076

RESUMO

BACKGROUND: Pathogen whole genome sequencing (WGS) is being incorporated into public health surveillance and disease control systems worldwide and has the potential to make significant contributions to infectious disease surveillance, outbreak investigation and infection prevention and control. However, to date, there are limited data regarding (i) the optimal models for integration of genomic data into epidemiological investigations and (ii) how to quantify and evaluate public health impacts resulting from genomic epidemiological investigations. METHODS: We developed the Pathogen Genomics in Public HeAlth Surveillance Evaluation (PG-PHASE) Framework to guide examination of the use of WGS in public health surveillance and disease control. We illustrate the use of this framework with three pathogens as case studies: Listeria monocytogenes, Mycobacterium tuberculosis and SARS-CoV-2. RESULTS: The framework utilises an adaptable whole-of-system approach towards understanding how interconnected elements in the public health application of pathogen genomics contribute to public health processes and outcomes. The three phases of the PG-PHASE Framework are designed to support understanding of WGS laboratory processes, analysis, reporting and data sharing, and how genomic data are utilised in public health practice across all stages, from the decision to send an isolate or sample for sequencing to the use of sequence data in public health surveillance, investigation and decision-making. Importantly, the phases can be used separately or in conjunction, depending on the need of the evaluator. Subsequent to conducting evaluation underpinned by the framework, avenues may be developed for strategic investment or interventions to improve utilisation of whole genome sequencing. CONCLUSIONS: Comprehensive evaluation is critical to support health departments, public health laboratories and other stakeholders to successfully incorporate microbial genomics into public health practice. The PG-PHASE Framework aims to assist public health laboratories, health departments and authorities who are either considering transitioning to whole genome sequencing or intending to assess the integration of WGS in public health practice, including the capacity to detect and respond to outbreaks and associated costs, challenges and facilitators in the utilisation of microbial genomics and public health impacts.


Assuntos
Ciência da Implementação , Infecções/diagnóstico , Listeria monocytogenes/isolamento & purificação , Mycobacterium tuberculosis/isolamento & purificação , SARS-CoV-2/isolamento & purificação , Sequenciamento Completo do Genoma/métodos , Genoma Bacteriano , Genoma Viral , Humanos , Infecções/epidemiologia , Listeria monocytogenes/genética , Mycobacterium tuberculosis/genética , Vigilância da População , Saúde Pública , SARS-CoV-2/genética
13.
Nat Commun ; 12(1): 4044, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34193872

RESUMO

What determines the rate (µ) and molecular spectrum of mutation is a fundamental question. The prevailing hypothesis asserts that natural selection against deleterious mutations has pushed µ to the minimum achievable in the presence of genetic drift, or the drift barrier. Here we show that, contrasting this hypothesis, µ substantially exceeds the drift barrier in diverse organisms. Random mutation accumulation (MA) in yeast frequently reduces µ, and deleting the newly discovered mutator gene PSP2 nearly halves µ. These results, along with a comparison between the MA and natural yeast strains, demonstrate that µ is maintained above the drift barrier by stabilizing selection. Similar comparisons show that the mutation spectrum such as the universal AT mutational bias is not intrinsic but has been selectively preserved. These findings blur the separation of mutation from selection as distinct evolutionary forces but open the door to alleviating mutagenesis in various organisms by genome editing.


Assuntos
Modelos Genéticos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Deriva Genética , Acúmulo de Mutações , Taxa de Mutação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Seleção Genética , Sequenciamento Completo do Genoma/métodos
14.
Sci Rep ; 11(1): 14558, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34267263

RESUMO

Whereas accelerated attention beclouded early stages of the coronavirus spread, knowledge of actual pathogenicity and origin of possible sub-strains remained unclear. By harvesting the Global initiative on Sharing All Influenza Data (GISAID) database ( https://www.gisaid.org/ ), between December 2019 and January 15, 2021, a total of 8864 human SARS-CoV-2 complete genome sequences processed by gender, across 6 continents (88 countries) of the world, Antarctica exempt, were analyzed. We hypothesized that data speak for itself and can discern true and explainable patterns of the disease. Identical genome diversity and pattern correlates analysis performed using a hybrid of biotechnology and machine learning methods corroborate the emergence of inter- and intra- SARS-CoV-2 sub-strains transmission and sustain an increase in sub-strains within the various continents, with nucleotide mutations dynamically varying between individuals in close association with the virus as it adapts to its host/environment. Interestingly, some viral sub-strain patterns progressively transformed into new sub-strain clusters indicating varying amino acid, and strong nucleotide association derived from same lineage. A novel cognitive approach to knowledge mining helped the discovery of transmission routes and seamless contact tracing protocol. Our classification results were better than state-of-the-art methods, indicating a more robust system for predicting emerging or new viral sub-strain(s). The results therefore offer explanations for the growing concerns about the virus and its next wave(s). A future direction of this work is a defuzzification of confusable pattern clusters for precise intra-country SARS-CoV-2 sub-strains analytics.


Assuntos
COVID-19/virologia , SARS-CoV-2/genética , Análise de Sequência de DNA/métodos , COVID-19/epidemiologia , COVID-19/transmissão , Biologia Computacional/métodos , DNA Viral/genética , Bases de Dados Genéticas , Previsões/métodos , Genoma Viral , Humanos , Aprendizado de Máquina , Mutação , Filogenia , SARS-CoV-2/classificação , SARS-CoV-2/patogenicidade , Sequenciamento Completo do Genoma/métodos
15.
Methods Mol Biol ; 2314: 459-480, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235666

RESUMO

Whole-genome sequencing is a powerful, high-resolution tool that can be used to generate accurate data on bacterial population structure, phylogeography, and mutations associated with antimicrobial resistance. The ability to sequence pathogen genomes directly from clinical specimens, without the requirement for in vitro culturing, is attractive in terms of time- and labor-saving, especially in the case of slow growing pathogens, such as Mycobacterium tuberculosis. However, clinical samples typically contain too low levels of pathogen nucleic acid, plus relatively high levels of human and natural microbiota DNA/RNA, to make this a viable option. Using a combination of whole-genome enrichment and deep sequencing, which has been proven to be a nonmutagenic approach, we can capture all known variations found within M. tuberculosis genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of M. tuberculosis directly from clinical samples and has the potential to be adapted to other pathogens with a similar clonal nature.


Assuntos
Testes Diagnósticos de Rotina/métodos , Genoma Bacteriano , Mycobacterium tuberculosis/genética , Escarro/microbiologia , Tuberculose/diagnóstico , Sequenciamento Completo do Genoma/métodos , Humanos , Tuberculose/microbiologia
16.
Methods Mol Biol ; 2314: 399-457, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235665

RESUMO

Whole-genome sequencing (WGS) has shown immense value in enabling identification and characterization of bacterial taxa. This is particularly true for mycobacteria, where culture-based characterization becomes delayed by the inherently slow growth rate of these organisms. This chapter reviews the general techniques behind WGS and their optimization, existing techniques for species-level identification and the advantages of WGS for this purpose, and a variety of useful tools for the genomic characterization of mycobacterial strains.


Assuntos
DNA Bacteriano/análise , Genoma Bacteriano , Genômica/métodos , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Análise de Sequência de DNA/métodos , Sequenciamento Completo do Genoma/métodos , DNA Bacteriano/genética , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/isolamento & purificação
17.
Int J Mol Sci ; 22(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34281179

RESUMO

Dermatophilus congolensis is a bacterial pathogen mostly of ruminant livestock in the tropics/subtropics and certain temperate climate areas. It causes dermatophilosis, a skin disease that threatens food security by lowering animal productivity and compromising animal health and welfare. Since it is a prevalent infection in ruminants, dermatophilosis warrants more research. There is limited understanding of its pathogenicity, and as such, there is no registered vaccine against D. congolensis. To better understanding the genomics of D. congolensis, the primary aim of this work was to investigate this bacterium using whole-genome sequencing and bioinformatic analysis. D. congolensis is a high GC member of the Actinobacteria and encodes approximately 2527 genes. It has an open pan-genome, contains many potential virulence factors, secondary metabolites and encodes at least 23 housekeeping genes associated with antimicrobial susceptibility mechanisms and some isolates have an acquired antimicrobial resistance gene. Our isolates contain a single CRISPR array Cas type IE with classical 8 Cas genes. Although the isolates originate from the same geographical location there is some genomic diversity among them. In conclusion, we present the first detailed genomic study on D. congolensis, including the first observation of tet(Z), a tetracycline resistance-conferring gene.


Assuntos
Dermatophilus/efeitos dos fármacos , Dermatophilus/genética , Actinobacteria/genética , Animais , Antibacterianos/farmacologia , Bovinos , Doenças dos Bovinos/metabolismo , Biologia Computacional/métodos , Dermatophilus/metabolismo , Genoma Bacteriano , Infecções por Bactérias Gram-Positivas/genética , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Positivas/veterinária , Doenças dos Cavalos/microbiologia , Cavalos , Resistência a Tetraciclina/genética , Sequenciamento Completo do Genoma/métodos
18.
Emerg Microbes Infect ; 10(1): 1293-1299, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34125658

RESUMO

The SARS-CoV-2 B.1.1.7 lineage is highly infectious and as of April 2021 accounted for 92% of COVID-19 cases in Europe and 59% of COVID-19 cases in the U.S. It is defined by the N501Y mutation in the receptor-binding domain (RBD) of the Spike (S) protein, and a few other mutations. These include two mutations in the N terminal domain (NTD) of the S protein, HV69-70del and Y144del (also known as Y145del due to the presence of tyrosine at both positions). We recently identified several emerging SARS-CoV-2 variants of concerns, characterized by Membrane (M) protein mutations, including I82T and V70L. We now identify a sub-lineage of B.1.1.7 that emerged through sequential acquisitions of M:V70L in November 2020 followed by a novel S:D178H mutation first observed in early February 2021. The percentage of B.1.1.7 isolates in the US that belong to this sub-lineage increased from 0.15% in February 2021 to 1.8% in April 2021. To date, this sub-lineage appears to be U.S.-specific with reported cases in 31 states, including Hawaii. As of April 2021, it constituted 36.8% of all B.1.1.7 isolates in Washington. Phylogenetic analysis and transmission inference with Nextstrain suggest this sub-lineage likely originated in either California or Washington. Structural analysis revealed that the S:D178H mutation is in the NTD of the S protein and close to two other signature mutations of B.1.1.7, HV69-70del and Y144del. It is surface exposed and may alter NTD tertiary configuration or accessibility, and thus has the potential to affect neutralization by NTD directed antibodies.


Assuntos
Mutação , SARS-CoV-2/classificação , Glicoproteína da Espícula de Coronavírus/genética , Proteínas da Matriz Viral/genética , Sequenciamento Completo do Genoma/métodos , Sítios de Ligação , Humanos , Modelos Moleculares , Filogenia , Domínios Proteicos , Estrutura Terciária de Proteína , SARS-CoV-2/genética , Análise de Sequência de RNA , Glicoproteína da Espícula de Coronavírus/química , Estados Unidos
20.
Int J Biol Macromol ; 184: 1035-1061, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34174315

RESUMO

Calmodulin (CaM) is considered as the most significant Ca2+ signaling messenger that mediate various biochemical and physiological reactions. IQ domain (IQD) proteins are plant specific CML/CaM calcium binding which are characterized by domains of 67 amino acids. 50, 50, 94, and 99 IQD genes were detected from G. arboreum (A2), G. raimondii (D5), G. barbadense (AD2) and G. hirsutum (AD1) respectively. Existence of more orthologous genes in cotton species than Arabidopsis, advocated that polyploidization produced new cotton specific orthologous gene clusters. Duplication of gene events depicts that IQD gene family of cotton evolution was under strong purifying selection. G. hirsutum exhibited high level synteny. GarIQD25 exhibited high expression in stem, root, flower, ovule and fiber in G. arboreum. In G. raimondii, GraIQD03 demonstrated upregulation across stem, ovule, fiber and seed. GbaIQD11 and GbaIQD62 exhibited upregulation in fiber development in G. barbadense. GhiIQD69 recognized as main candidate genes for plant parts, floral tissues, fiber and ovule development. Promotor analysis identified cis-regulatory elements were involved in plant growth and development. Overwhelmingly, present study paves the way to better understand the evolution of cotton IQD genes and lays a foundation for future investigation of IQD in cotton.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Gossypium/genética , Sequenciamento Completo do Genoma/métodos , Mapeamento Cromossômico , Diploide , Regulação da Expressão Gênica de Plantas , Gossypium/classificação , Família Multigênica , Filogenia , Proteínas de Plantas/genética , Seleção Genética , Tetraploidia , Distribuição Tecidual
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...