Characterization of 23S rRNA domain V mutations in gastric biopsy patients from the eastern Amazon

Resistance of Helicobacter pylori to clarithromycin is characterised by simple point mutations in the 23S ribosomal RNA (rRNA) gene and is responsible for the majority of cases of failure to eradicate this bacterium. In this paper, we characterised the variability of the 23S rRNA gene in biopsies of patients with gastric pathologies in the eastern Amazon (Northern Region of Brazil) using PCR and sequencing. A total of 49 sequences of H. pylori strains were analysed and of those, 75.6% presented nucleotide substitutions: A2142G (3.3%), T2182C (12.9%), G2224A (6.45%), T2215C (61.3%), A2192G (3.3%), G2204C (6.4%) and T2221C (6.4%). Of the mutations identified, four are known mutations related to cases of resistance and 16.1% are not yet described, revealing a high prevalence of mutations in the H. pylori 23S rRNA gene among the strains circulating in the in the eastern Amazon. The high prevalence in individuals with gastric pathologies in the Northern Region of Brazil demonstrates the need for characterising the profile of these strains to provide correct therapy for patients, considering that mutations in this gene are normally associated with resistance to the primary medication used in controlling H. pylori infection.

Filogenia molecular dos xenarthra (Mammalia): análise do grupo cingulata a partir de sequências nucleotídicas do gene mitocondrial rRNA 16S

Os Xenarthra são o grupo de mamíferos que inclui os tatus, os tamanduás e as preguiças. A América do Sul serviu de cenário para a história natural do grupo que, somente no fim do Cenozóico, dispersou-se para a América Central e, com uma perda de variedade, chegou à América do Norte e à algu-mas ilhas do Caribe. Trinta e uma espécies estão descritas dentro da linha-gem dos Xenarthra. Elas estão classificadas em 13 gêneros, quatro famílias (Bradypodidae, Megalonychidae, Myrmecophagidae e Dasypodidae) e duas ordens (Cingulata e Pilosa). A filogenia deste grupo tem sido alvo de diver-sas pesquisas que analisaram tanto dados morfológicos, quanto moleculares. Delsuc et al. (2003) analisaram seqüências de genes mitocondriais e nucleares e confirmaram a monofilia das três subfamílias (Dasypodinae, Euphacti-nae e Tolypeutinae) inclusas na família Dasypodidae. Delsuc et al. (2003) geraram a seguinte árvore: (((Bradypus, Choloepus)100, ((Myrmecophaga, Tamandua)100, Cyclopes)100), ((D. kappleri, D. novemcinctus)100, (Toly-pentes, (Priodontes, Cabassous)54)100, (Zaedyus, (Euphractus, Chaetophrac-tus)60)100)). Gaudin (2005) apresentou um trabalho que reviu e ampliou as análises morfológicas apresentadas até então, concluindo que os tatus atu-ais estão divididos em dois grupos, um mais basal (Dasypodinae) e outro mais derivado (Euphractinae), de acordo com o seguinte arranjo: (Bradypus, Tamandua), (Dasypus, (Priodontes, (Cabassous, (Tolypeutes, (Euphractus, Chaetophractus, (Zaedyus, Chlamyphorus)42)36)72)72)40)85). Neste traba-lho utilizou-se parte do gene mitocondrial rRNA 16S de 12 táxons atu-ais de Xenarthra para analisar a filogenia do grupo através do critério de máxima verossimilhança. Nossos resultados são apresentados analisando-se o gene 16S e analisando o banco de dados do 16S mais o de Delsuc et al. (2003). Nas duas situações, as filogenias apresentadas apóiam os resulta-dos de Delsuc et al. (2003): (Bradypus, (Choloepus, ((Cyclopes, (Myrme-cophaga, Tamandua)100)100, (Dasypus, (((Cabassous, Priodontes)68, Toly-peutes)100,((Chaetophractus, Euphractus)65, Zaedyus)100)100)100)100)100). Uma melhora nos valores de bootstrap nos ramos dentro das sub-famílias da família Dasypodidae é percebida em relação ao trabalho de Delsuc et al. (2003). Acreditamos que Elementos de Transposição do tipo (LINES) são os marcadores moleculares mais adequados para confirmar o arranjo obtido com as seqüências de genes mitocondriais e nucleares.

Comparative Chromosome Mapping of U2 snRNA and 5S rRNA Genes in Gymnotus Species (Gymnotiformes, Gymnotidae): Evolutionary Dynamics and Sex Chromosome Linkage in G. pantanal

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Analise microbiológica da flora intestinal do mosquito Aedes Aegypti através do sequenciamento do gene 16S rRNA na plataforma Illumina MiSeq

Dengue é uma arbovirose que afeta cerca de 100 milhões de pessoas anualmente, em mais de 100 países situados nas regiões tropicais e subtropicais. Foi considerada a doença viral que mais cresceu no ultimo ano, repercutindo em impactos sociais e econômicos nas regiões endêmicas devido às altas taxas de morbidade e mortalidade desencadeadas pela infecção. O principal vetor da dengue é o mosquito Aedes aegypti, presente em toda a faixa tropical e subtropical. Por apresentar hematofagia antropofílica, rápido desenvolvimento e características comportamentais especificas, é um excelente transmissor do vírus dengue. Medidas de controle da disseminação da dengue são restritas à eliminação do mosquito vetor, e um tratamento específico ainda não foi desenvolvido, bem como a criação de uma vacina que previna simultaneamente a infecção pelos quatro sorotipos do arbovírus. Uma característica que determina a disseminação de doenças é a alta competência vetorial de seus mosquitos transmissores, que tem sido associada à composição da microbiota intestinal do inseto. As bactérias presente no intestino do mosquito exercem funções relacionadas a sua nutrição, desenvolvimento e reprodução, e são também um importante fator na eliminação de patógenos, por interferirem diretamente na atividade viral, ou indiretamente a partir da ativação das vias antivirais pelos micro-organismos. Dessa forma, este trabalho visa estudar a diversidade microbiana intestinal do mosquito Aedes aegypti em diferentes estágios de vida, através de sequenciamento de última geração com a plataforma MiSeq Illumina

A phylogenetic analysis of Brycon and Henochilus (Characiformes, Characidae, Bryconinae) based on the mitochondrial gene 16S rRNA

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

The addition of a capped mini-exon [spliced leader (SL)] through trans-splicing is essential for the maturation of RNA polymerase (pol) II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA) are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS) region. Additionally, we detected the SL-5' ETS molecule using three distinct methods and located the acceptor site between two known 5' ETS rRNA processing sites (A' and A1) in four different trypanosomatids. Moreover, we detected a polyadenylated 5' ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin), we observed SL-5' ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA) led to the accumulation of SL-5' ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

Analysis of 16S rRNA and mxaF genes revealing insights into Methylobacterium niche-specific plant association

The genus Methylobacterium comprises pink-pigmented facultative methylotrophic (PPFM) bacteria, known to be an important plant-associated bacterial group. Species of this group, described as plant-nodulating, have the dual capacity of producing cytokinin and enzymes, such as pectinase and cellulase, involved in systemic resistance induction and nitrogen fixation under specific plant environmental conditions. The aim hereby was to evaluate the phylogenetic distribution of Methylobacterium spp. isolates from different host plants. Thus, a comparative analysis between sequences from structural (16S rRNA) and functional mxaF (which codifies for a subunit of the enzyme methanol dehydrogenase) ubiquitous genes, was undertaken. Notably, some Methylobacterium spp. isolates are generalists through colonizing more than one host plant, whereas others are exclusively found in certain specific plant-species. Congruency between phylogeny and specific host inhabitance was higher in the mxaF gene than in the 16S rRNA, a possible indication of function-based selection in this niche. Therefore, in a first stage, plant colonization by Methylobacterium spp. could represent generalist behavior, possibly related to microbial competition and adaptation to a plant environment. Otherwise, niche-specific colonization is apparently impelled by the host plant.

Identification of SL addition trans-splicing acceptor sites in the internal transcribed spacer I region of pre-rRNA in Leishmania (Leishmania) amazonensis

Trypanosomatidae is a family of early branching eukaryotes harbouring a distinctive repertoire of gene expression strategies. Functional mature messenger RNA is generated via the trans-splicing and polyadenylation processing of constitutively transcribed polycistronic units. Recently, trans-splicing of pre-small subunit ribosomal RNA in the 5' external transcribed spacer region and of precursor tRNAsec have been described. Here, we used a previously validated semi-nested reverse transcription-polymerase chain reaction strategy to investigate internal transcribed spacer (ITS) I acceptor sites in total RNA from Leishmania (Leishmania) amazonensis. Two distinct spliced leader-containing RNAs were detected indicating that trans-splicing reactions occur at two AG acceptor sites mapped in this ITS region. These data provide further evidence of the wide spectrum of RNA molecules that act as trans-splicing acceptors in trypanosomatids.

Analysis of 16S rRNA and mxaF genes reveling insights into Methylobacterium niche-specific plant association

The genus Methylobacterium comprises pink-pigmented facultative methylotrophic (PPFM) bacteria, known to be an important plant-associated bacterial group. Species of this group, described as plant-nodulating, have the dual capacity of producing cytokinin and enzymes, such as pectinase and cellulase, involved in systemic resistance induction and nitrogen fixation under specific plant environmental conditions. The aim hereby was to evaluate the phylogenetic distribution of Methylobacterium spp. isolates from different host plants. Thus, a comparative analysis between sequences from structural (16S rRNA) and functional mxaF (which codifies for a subunit of the enzyme methanol dehydrogenase) ubiquitous genes, was undertaken. Notably, some Methylobacterium spp. isolates are generalists through colonizing more than one host plant, whereas others are exclusively found in certain specific plant-species. Congruency between phylogeny and specific host inhabitance was higher in the mxaF gene than in the 16S rRNA, a possible indication of function-based selection in this niche. Therefore, in a first stage, plant colonization by Methylobacterium spp. could represent generalist behavior, possibly related to microbial competition and adaptation to a plant environment. Otherwise, niche-specific colonization is apparently impelled by the host plant.

16S rRNA terminal restriction fragment length polymorphism for the characterization of the nasopharyngeal microbiota

A novel non-culture based 16S rRNA Terminal Restriction Fragment Length Polymorphism (T-RFLP) method using the restriction enzymes Tsp509I and Hpy166II was developed for the characterization of the nasopharyngeal microbiota and validated using recently published 454 pyrosequencing data. 16S rRNA gene T-RFLP for 153 clinical nasopharyngeal samples from infants with acute otitis media (AOM) revealed 5 Tsp509I and 6 Hpy166II terminal fragments (TFs) with a prevalence of >10%. Cloning and sequencing identified all TFs with a prevalence >6% allowing a sufficient description of bacterial community changes for the most important bacterial taxa. The conjugated 7-valent pneumococcal polysaccharide vaccine (PCV-7) and prior antibiotic exposure had significant effects on the bacterial composition in an additive main effects and multiplicative interaction model (AMMI) in concordance with the 16S rRNA 454 pyrosequencing data. In addition, the presented T-RFLP method is able to discriminate S. pneumoniae from other members of the Mitis group of streptococci, which therefore allows the identification of one of the most important human respiratory tract pathogens. This is usually not achieved by current high throughput sequencing protocols. In conclusion, the presented 16S rRNA gene T-RFLP method is a highly robust, easy to handle and a cheap alternative to the computationally demanding next-generation sequencing analysis. In case a lot of nasopharyngeal samples have to be characterized, it is suggested to first perform 16S rRNA T-RFLP and only use next generation sequencing if the T-RFLP nasopharyngeal patterns differ or show unknown TFs.

Real-time quantitative broad-range PCR assay for detection of the 16S rRNA gene followed by sequencing for species identification

Here we determined the analytical sensitivities of broad-range real-time PCR-based assays employing one of three different genomic DNA extraction protocols in combination with one of three different primer pairs targeting the 16S rRNA gene to detect a panel of 22 bacterial species. DNA extraction protocol III, using lysozyme, lysostaphin, and proteinase K, followed by PCR with the primer pair Bak11W/Bak2, giving amplicons of 796 bp in length, showed the best overall sensitivity, detecting DNA of 82% of the strains investigated at concentrations of < or =10(2) CFU in water per reaction. DNA extraction protocols I and II, using less enzyme treatment, combined with other primer pairs giving shorter amplicons of 466 bp and 342 or 346 bp, respectively, were slightly more sensitive for the detection of gram-negative but less sensitive for the detection of gram-positive bacteria. The obstacle of detecting background DNA in blood samples spiked with bacteria was circumvented by introducing a broad-range hybridization probe, and this preserved the minimal detection limits observed in samples devoid of blood. Finally, sequencing of the amplicons generated using the primer pair Bak11W/Bak2 allowed species identification of the detected bacterial DNA. Thus, broad-spectrum PCR targeting the 16S rRNA gene in the quantitative real-time format can achieve an analytical sensitivity of 1 to 10 CFU per reaction in water, avoid detection of background DNA with the introduction of a broad-range probe, and generate amplicons that allow species identification of the detected bacterial DNA by sequencing. These prerequisites are important for its application to blood-containing patient samples.