RESUMO
The RNA-based study provides an excellent indication of an organism's gene expression profile. Obtaining high-yield and high-purity RNA from Gram-positive and acid-fast bacteria is difficult without high-end kits and facilities. We optimised effective and simple protocol for RNA isolation that is a combination of enzymatic, physical and chemical treatment to disrupt cells. We successfully isolated high quality intact total RNA with yields ranging from 23.13 ± 0.40 to 61.51 ± 0.27 µg and the 260/280 purity ratio of 1.95 ± 0.01 to 2.05 ± 0.01 from Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Mycobacterium smegmatis. These results represents a significantly enhanced yield and purity compared to other combination of techniques which we performed. Compared to previous studies the yield obtained by this method is high for the studied organisms. Furthermore the yielded RNA was successfully used for downstream applications such as quantitative real time PCR. The described method can be easily optimised and used for various bacteria.
Assuntos
RNA Bacteriano , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/isolamento & purificação , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/isolamento & purificação , Enterococcus faecalis/genética , Enterococcus faecalis/isolamento & purificação , Mycobacterium smegmatis/genéticaRESUMO
BACKGROUND: The objective of this study was to describe the bacterial communities associated with pediatric patients with endodontic infections of temporal teeth by targeting the 16S rRNA gene using pyrosequencing. MATERIAL AND METHODS: Microbiological samples were obtained from the lower primary molars of thirteen 13 pediatric patients with dental infections. An aspiration method for microbiological sampling was used. The identification of microbiota employing the pyrosequencing method by targeting the 16S gene was performed. RESULTS: Ribosomal 16S RNA gene sequences were amplified, obtaining a total of 16,182 sequences from 13 primary infected molars (13 different individuals) by pyrosequencing. Bacteroidetes phyla (35.15%) were the most abundant followed by Firmicutes (33.3%) and Fusobacteria (10.05%); the presence of specific pathogenic bacteria was determined as well. CONCLUSIONS: The infected root canal of primary teeth contains a high diversity of anaerobic bacteria, and Bacteroidetes, Firmicutes, and Fusobacteria phyla were the most abundant; Prevotella and Streptococcus genera were the most prevalent
No disponible
Assuntos
Humanos , Masculino , Feminino , Pré-Escolar , Criança , Dente Decíduo/microbiologia , Cavidade Pulpar/metabolismo , Bactérias/isolamento & purificação , Doenças Periapicais/microbiologia , Sequenciamento de Nucleotídeos em Larga Escala , RNA Bacteriano/isolamento & purificação , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/isolamento & purificação , MéxicoRESUMO
BACKGROUND: The objective of this cross-sectional clinical study was to analyze the differences in the microbiome in gingival sulci of adult patients in the presence or absence of chronic periodontitis. MATERIAL AND METHODS: Patients with or without periodontal disease were included in this cross-sectional study. Subgingival biofilm samples were collected and analyzed by 16S massive pyrosequencing. Functional analyses were also performed. RESULTS: A total of 15 phyla, 154 genera and 351 species were detected globally. Differences between disease and non-disease samples were observed in all taxonomical levels which suggest functional profile changes in the community. It was found that the main species associated with non-disease samples were reduced in disease but not completely suppressed. Analysis of the functional potential of the biofilms revealed a significantly higher activity related to endocytosis and phosphatidylinositol signaling in the disease group but lower cell adhesion molecules. CONCLUSIONS: Specific differences between health and disease suggest functional profile changes in the community, although bacteria associated with periodontal disease are also increased in health. Transcriptome studies should be conducted to confirm and deepen metabolic dysfunctions
No disponible
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Idoso , Periodontite Crônica/microbiologia , Gengiva/microbiologia , Bactérias/isolamento & purificação , Estudos de Casos e Controles , Estudos Transversais , Sequenciamento de Nucleotídeos em Larga Escala , Microbiota , Reação em Cadeia da Polimerase , Bactérias/genética , RNA Ribossômico 16S/genética , Estatísticas não Paramétricas , RNA Bacteriano/isolamento & purificaçãoRESUMO
Abstract Objective The rDNA-based method is unable to distinguish between alive and dead cells. Alternatively, bacterial viability can be assessed by molecular methods based on ribosomal RNA (rRNA). Therefore, this study aimed to detect viable streptococci in root canal samples using rRNA-based reverse transcription polymerase chain reaction (RT-PCR), compared to an rDNA-based PCR assay. Methodology Microbiological root canal samples were obtained from 32 teeth with primary endodontic infections before (S1) and after chemomechanical preparation (S2), and after removal of intracanal medication (S3). RNA and DNA were extracted, and complementary DNA (cDNA) was synthesized from RNA using RT reaction. cDNA and genomic DNA were subjected to PCR with primers complementary to the 16S rRNA sequences of Streptococcus spp. McNemar's test was used to compare the detection rate of both assays (P<0.05). Results Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using rRNA- and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA-based RT-PCR, compared to 15.62% (5/32) using rDNA-based PCR. Finally, in S3 samples, streptococci were not detected by rRNA, whereas rDNA-based PCR still detected the bacteria in 12.5% (4/32) of the samples. The total number of PCR-positive reactions in the rDNA-based PCR was higher than in the rRNA-based assay (P<0.05). Conclusions The rRNA-based RT-PCR showed a lower detection rate of streptococci when compared to the rDNA-based PCR, suggesting that the latter may have detected dead cells of streptococci in root canal samples.
Assuntos
Humanos , Streptococcus/isolamento & purificação , DNA Ribossômico/isolamento & purificação , RNA Ribossômico/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Cavidade Pulpar/microbiologia , Tratamento do Canal Radicular/métodos , Streptococcus/genética , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/genética , DNA Ribossômico/genética , RNA Bacteriano/isolamento & purificação , RNA Bacteriano/genética , RNA Ribossômico/genética , Reprodutibilidade dos TestesRESUMO
Biofilm formed by Staphylococcus aureus is considered an important virulence trait in the pathogenesis of infections associated with implantable medical devices. Gene expression analyses are important strategies for determining the mechanisms involved in production and regulation of biofilm. Obtaining intact RNA preparations is the first and most critical step for these studies. In this article, we describe an optimized protocol for obtaining total RNA from sessile cells of S. aureus using the RNeasy Mini Kit. This method essentially consists of a few steps, as follows: 1) addition of acetone-ethanol to sessile cells, 2) lysis with lysostaphin at 37°C/10 min, 3) vigorous mixing, 4) three cycles of freezing and thawing, and 5) purification of the lysate in the RNeasy column. This simple pre-kit procedure yields high-quality total RNA from planktonic and sessile cells of S. aureus.
Assuntos
Técnicas Bacteriológicas/normas , Biofilmes/crescimento & desenvolvimento , RNA Bacteriano/isolamento & purificação , Staphylococcus aureus/genética , Técnicas Bacteriológicas/métodos , Eletroforese em Gel de Ágar , Proteínas Hemolisinas/metabolismo , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Controle de Qualidade , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Reversa , Staphylococcus aureus/fisiologiaRESUMO
Poribacterial clone libraries constructed for Aplysina fulva sponge specimens were analysed with respect to diversity and phylogeny. Results imply the coexistence of several, prevalently "intraspecific" poribacterial genotypes in a single sponge host, and suggest quantitative analysis as a desirable approach in studies of the diversity and distribution of poribacterial cohorts in marine sponges
Assuntos
Microbiologia Ambiental , Variação Genética , Técnicas In Vitro , Filogenia , Poríferos , RNA Bacteriano/isolamento & purificação , Genótipo , Métodos , Estudos de Avaliação como AssuntoRESUMO
Mycobacterium tuberculosis gene expression studies have involved "in vitro", "ex vivo" and "in vivo" experiments (animal models), but without the expected success. We propose that key features of human tuberculosis could be discovered by studying the M. tuberculosis gene expression within the human host. Therefore, we isolated totalM. tuberculosis mRNA from human clinical respiratory specimens of patients diagnosed with pulmonary tuberculosis; after this, we synthesized the dscDNA and tested it by qualitative RT-PCR assays. We detected the expression of IS6110 insertion sequence and of the "housekeeping" genes 16SrRNA andsigA in M. tuberculosis grown in vivo (pulmonary tuberculosis) as well as grown in vitro M. tuberculosis. mprA and mprB genes expression, which code the MprAB signal transduction system, were only detected in M. tuberculosis grown in vitro. Our results provide the first step towards a non invasive method for the study of the transcriptome of M. tuberculosis within its native host, to analyze "in vivo" regulation of the genetic determinants required for virulence and pathogenesis.
El estudio de la expresión génica de Mycobacterium tuberculosis ha involucrado la experimentación "in vitro ", "ex vivo " e "in vivo " (modelos animales), pero aún sin el éxito esperado. Proponemos que revelar los factores clave de la tuberculosis humana requiere investigar la expresión génica de M. tuberculosis dentro del ser humano ("in vivo "). Para ello, aislamos el mRNA total de M. tuberculosis, desde muestras clínicas respiratorias de pacientes con diagnóstico de tuberculosis pulmonar; posteriormente, sintetizamos el dscDNA y lo analizamos mediante RT-PCR cualitativo. Detectamos la expresión de la secuencia de inserción IS6110 y de los genes "housekeeping " 16SrRNA y sigA en M. tuberculosis creciendo in vivo (tuberculosis pulmonar) así como cultivado in vitro. La expresión de los genes mprA y mprB, que codifican el sistema de transducción de señales MprAB, sólo se detectó en M. tuberculosis crecido in vitro. Con nuestros resultados damos el primer paso hacia la implementación de un método no invasivo para el estudio del transcriptoma de M. tuberculosis, dentro de su único hospedero natural, con el fin de analizar la regulación "in vivo" de los determinantes genéticos requeridos para su virulencia y patogénesis.
Assuntos
Humanos , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/microbiologia , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , RNA Bacteriano/genética , RNA Mensageiro/genética , Escarro/microbiologia , Expressão Gênica , Mycobacterium tuberculosis/isolamento & purificação , Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase em Tempo Real , Técnicas de Amplificação de Ácido Nucleico , Técnicas de CulturaRESUMO
Fusobacterium nucleatum é uma espécie bacteriana Gram-negativa, anaeróbia estrita e uma das espécies frequentemente encontradas nas infecções primárias do sistema de canais radiculares. Esta espécie tem grande importância na formação de biofilmes por ser uma ponte de união entre espécies que não são capazes de interagir. Os micro-organismos constituintes de biofilmes trocam material genético, aumentando a tolerancia dos mesmos e é quase impossível um isolado clínico ter os genes totalmente iguais à cepa padrão de coleções de cultura como da ATCC (American Type Culture Colection). O presente estudo investigou a espécie bacteriana anaeróbia Fusobacterium nucleatum isolada de canais radiculares, comparando-a com sua cepa de referência. Foi feito a comparação da suscetibilidade microbiana in vitro por meio de cultura microbiológica pelo método do E-test, com as cepas em crescimento planctônico e em biofilme. Também foi definido um protocolo de Purificação de RNA para esta espécie em ambas as condições de crescimento. As cepas clínicas de F. nucelatum foram isoladas por meio da cultura microbiológica de 23 pacientes que apresentavam dentes com infecção primária e periodontite apical visível em radiografia. Foi feito isolamento e identificação da espécie por série bioquímica com testes comerciais (Sistema Api, Bio-Meriéux, França) e PCR convencional, sendo no total 4 isolados clínicos investigados. Foi verificada a suscetibilidade antimicrobiana dos seguintes antibióticos: Amoxicilina, Amoxicilina + ácido clavulânico, Ampicilina, Azitromicina, Clindamicina, Eritromicina e Metronidazol. O protocolo para purificação de RNA foi feito com microesferas de zircônia, dispositivo bead beater, kit comercial RNeasy (Qiagen) e transcrição para DNA complementar (cDNA). A qualidade da purificação foi testada quanto a sua capacidade de amplificação pela reação em cadeia da polimerase em tempo real (qPCR) utilizando primer para o gene 16s RNA específico para F. nucelatum...
Fusobacterium nucleatum is a Gram-negative bacterial species, strict anaerobic and one of the species often found in primary infection of the root canal system. This species has great importance in biofilm formation to be a union bridge between species which are not able to act alone. The constituent microorganisms of the biofilm exchange each tother genetic material, increasing the strength of them. It is almost impossible for a clinical isolate have genes totally equal to a standard strain, such as strains of culture collections like ATCC (American Type Culture Collection). The present study investigated anaerobic bacterial species Fusobacterium nucleatum isolated from root canals, comparing them to the ATCC strain. The microbial in vitro susceptibility of biofilm and planktonic growth of the strains was compared by means of microbiological culture and the E-test method, with the antibiotics Amoxicillin, Amoxicillin + clavulanic acid, Ampicillin, Azithromycin, Clindamycin, Eritromycin and Metronidazole.. Also, a RNA Purification protocol for the strains under the same growth conditions was defined. Clinical isolates were obtained by microbiological samples of patients with teeth with pulp necrosis and apical periodontitis visible on radiographs. The species isolation and identification were performed using commercial biochemical tests (Sistema Api, Bio-Meriéux, France) and conventional PCR, obtaining four clinical isolates. The protocol for RNA purification was done with zirconia beads, bead beater device and commercial kit RNeasy (Qiagen) and transcribed into complementary DNA (cDNA). The quality of purification was tested for its ability of amplification by real time polymerase chain reaction (qPCR) using primer for the gene 16s RNA specific for F. nucleatum. All tested trains were 100% susceptible to amoxicillin + clavulanic acid, ampicillin, azithromycin, clindamycin and metronidazole. Both types of bacterial growth showed resistance to...
Assuntos
Humanos , Fusobacterium nucleatum/genética , Fusobacterium nucleatum/isolamento & purificação , Expressão Gênica , RNA Bacteriano/isolamento & purificação , Antibacterianos/farmacologia , Biofilmes/crescimento & desenvolvimento , Cavidade Pulpar/microbiologia , Fusobacterium nucleatum , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase em Tempo Real , Fatores de TempoRESUMO
Nucleic-acid based methods for bacterial identification are extremely useful in diagnostic applications due to their specificity and sensitivity. However, they require an optimal purification of the target molecules. As part of the development of a new diagnostic method for the detection of bacterial RNA in cow milk, we have compared four commercially available RNA extraction kits for the isolation of bacterial RNA from spiked UHT milk samples. The kits were compared in terms of extraction efficiency and RNA purity using two bacterial species, the Gram negative Escherichia coli and the Gram positive Staphylococcus aureus. Two kits are based in silica-matrix extraction, and the other two in the guanidinium thiocyanate-phenol-chloroform extraction. In our hands, the RNeasy Protect Bacteria Mini kit from QIAGEN was the best performing in terms of RNA yield, quality, reproducibility and consumable needs, under the conditions here described.
Assuntos
RNA Bacteriano/isolamento & purificação , Escherichia coli/isolamento & purificação , Leite/microbiologia , Staphylococcus aureus/isolamento & purificaçãoRESUMO
The presence of extracellular polysaccharides matrix makes extraction and purification of RNA from Streptococcus mutans within biofilms challenging. In this study, several approaches to purify RNA extracted from S. mutans in suspension cultures and biofilms were examined. The combination of sonication (3 pulses of 30 s at 7 W), suspension in NAES buffer (50 mM sodium acetate buffer, 10 mM EDTA and 1 percent SDS; pH 5.0) and homogenization-mechanical cells disruption in NAES- acid phenol:chloroform, yielded 9.04 mg (or 0.52 mg) of crude preparation of RNA per 100 mg of total cell (or biofilm) dry-weight. The crude RNA preparations were subjected to various DNAse I treatments. The combination of DNAse I in silica-gel based column followed by recombinant DNase I in solution provided the best genomic DNA removal, resulting in 4.35 mg (or 0.06 mg) of purified RNA per 100 mg of total cell (or biofilm) dry-weight. The cDNAs generated from the purified RNA sample were efficiently amplified using gtfB S. mutans-specific primers. The results showed a method that yields high-quality RNA from both planktonic cells and biofilms of S. mutans in sufficient quantity and quality for real-time RT-PCR analyses.