BackgroundThe study of microbial communities has been revolutionised in recent years by the widespread adoption of culture independent analytical techniques such as 16S rRNA gene sequencing and metagenomics. One potential confounder of these sequence-based approaches is the presence of contamination in DNA extraction kits and other laboratory reagents.ResultsIn this study we demonstrate that contaminating DNA is ubiquitous in commonly used DNA extraction kits and other laboratory reagents, varies greatly in composition between different kits and kit batches, and that this contamination critically impacts results obtained from samples containing a low microbial biomass. Contamination impacts both PCR-based 16S rRNA gene surveys and shotgun metagenomics. We provide an extensive list of potential contaminating genera, and guidelines on how to mitigate the effects of contamination.ConclusionsThese results suggest that caution should be advised when applying sequence-based techniques to the study of microbiota present in low biomass environments. Concurrent sequencing of negative control samples is strongly advised.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-014-0087-z) contains supplementary material, which is available to authorized users.
Evasion of clinical interventions by Streptococcus pneumoniae occurs through selection of non-susceptible genomic variants. Here we use genome sequencing of 3,085 pneumococcal carriage isolates from a 2.4 km2 refugee camp to enable unprecedented resolution of the process of recombination, and highlight its impact on population evolution. Genomic recombination hotspots show remarkable consistency between lineages, indicating common selective pressures acting at certain loci, particularly those associated with antibiotic resistance. Temporal changes in antibiotic consumption are reflected in changes in recombination trends demonstrating rapid spread of resistance when selective pressure is high. The highest frequencies of receipt and donation of recombined DNA fragments were observed in non-encapsulated lineages, implying that this largely overlooked pneumococcal group, which is beyond the reach of current vaccines, may play a major role in genetic exchange and adaptation of the species as a whole. These findings advance our understanding of pneumococcal population dynamics and provide important information for the design of future intervention strategies.
Traditional genetic association studies are very difficult in bacteria, as the generally limited recombination leads to large linked haplotype blocks, confounding the identification of causative variants. Beta-lactam antibiotic resistance in Streptococcus pneumoniae arises readily as the bacteria can quickly incorporate DNA fragments encompassing variants that make the transformed strains resistant. However, the causative mutations themselves are embedded within larger recombined blocks, and previous studies have only analysed a limited number of isolates, leading to the description of “mosaic genes” as being responsible for resistance. By comparing a large number of genomes of beta-lactam susceptible and non-susceptible strains, the high frequency of recombination should break up these haplotype blocks and allow the use of genetic association approaches to identify individual causative variants. Here, we performed a genome-wide association study to identify single nucleotide polymorphisms (SNPs) and indels that could confer beta-lactam non-susceptibility using 3,085 Thai and 616 USA pneumococcal isolates as independent datasets for the variant discovery. The large sample sizes allowed us to narrow the source of beta-lactam non-susceptibility from long recombinant fragments down to much smaller loci comprised of discrete or linked SNPs. While some loci appear to be universal resistance determinants, contributing equally to non-susceptibility for at least two classes of beta-lactam antibiotics, some play a larger role in resistance to particular antibiotics. All of the identified loci have a highly non-uniform distribution in the populations. They are enriched not only in vaccine-targeted, but also non-vaccine-targeted lineages, which may raise clinical concerns. Identification of single nucleotide polymorphisms underlying resistance will be essential for future use of genome sequencing to predict antibiotic sensitivity in clinical microbiology.
BackgroundThe soil-transmitted helminth (STH), Trichuris trichiura colonises the human large intestine where it may modify inflammatory responses, an effect possibly mediated through alterations in the intestinal microbiota. We hypothesised that patent T. trichiura infections would be associated with altered faecal microbiota and that anthelmintic treatment would induce a microbiota resembling more closely that observed in uninfected individuals.Materials and MethodsSchool children in Ecuador were screened for STH infections and allocated to 3 groups: uninfected, T. trichiura only, and mixed infections with T. trichiura and Ascaris lumbricoides. A sample of uninfected children and those with T. trichiura infections only were given anthelmintic treatment. Bacterial community profiles in faecal samples were studied by 454 pyrosequencing of 16 S rRNA genes.ResultsMicrobiota analyses of faeces were done for 97 children: 30 were uninfected, 17 were infected with T. trichiura, and 50 with T. trichiura and A. lumbricoides. Post-treatment samples were analyzed for 14 children initially infected with T. trichiura alone and for 21 uninfected children. Treatment resulted in 100% cure of STH infections. Comparisons of the microbiota at different taxonomic levels showed no statistically significant differences in composition between uninfected children and those with T. trichiura infections. We observed a decreased proportional abundance of a few bacterial genera from the Clostridia class of Firmicutes and a reduced bacterial diversity among children with mixed infections compared to the other two groups, indicating a possible specific effect of A. lumbricoides infection. Anthelmintic treatment of children with T. trichiura did not alter faecal microbiota composition.DiscussionOur data indicate that patent human infections with T. trichiura may have no effect on faecal microbiota but that A. lumbricoides colonisation might be associated with a disturbed microbiota. Our results also catalogue the microbiota of rural Ecuadorians and indicate differences with individuals from more urban industrialised societies.
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