This study evaluated the microbial diversity and community structure of three different kefir grains collected in different regions of Brazil, by combining two cultureindependent methods: PCR-DGGE and barcode pyrosequencing. The DGGE analysis showed that the dominant bacterial populations in all three grains were similar and composed of two Lactobacillus species: Lactobacillus kefiranofaciens and Lactobacillus kefiri. The yeast community was dominated by Saccharomyces cerevisiae, which was present in all three samples. A total of 14,314 partial 16S rDNA sequence reads were obtained from the three grains by pyrosequencing. Sequence analysis grouped the reads into three phyla, of which Firmicutes was the most abundant. Members of the genus Lactobacillus were predominant operational taxonomic units (OTUs) in all samples, comprising up to 96% of the sequences. At low levels, OTUs belonging to other lactic-acid bacteria species and members of different phyla were also found. Two of the grains showed identical DGGE profiles and a similar number of OTUs, while the third sample showed the highest diversity by both techniques. The pyrosequencing approach allowed the identification of bacteria that were present in low numbers and are rarely associated with the microbial community of this complex ecosystem.

IntroductionBacteria present in the apical root canal system are directly involved with the pathogenesis of post-treatment apical periodontitis. This study used a next-generation sequencing approach to identify the bacterial taxa occurring in cryopulverized apical root samples from root canal-treated teeth with post-treatment disease.MethodsApical root specimens obtained during periradicular surgery of ten adequately treated teeth with persistent apical periodontitis were cryogenically ground. DNA was extracted from the powder and the microbiome was characterized on the basis of the V4 hypervariable region of the 16S rRNA gene by using paired-end sequencing on Illumina MiSeq device.ResultsAll samples were positive for the presence of bacterial DNA. Bacterial taxa were mapped to 11 phyla and 103 genera composed by 538 distinct operational taxonomic units (OTUs) at 3% of dissimilarity. Over 85% of the sequences belonged to 4 phyla: Proteobacteria, Firmicutes, Fusobacteria and Actinobacteria. In general, these 4 phyla accounted for approximately 80% of the distinct OTUs found in the apical root samples. Proteobacteria was the most abundant phylum in 6/10 samples. Fourteen genera had representatives identified in all cases. Overall, the genera Fusobacterium and Pseudomonas were the most dominant. Enterococcus was found in 4 cases, always in relatively low abundance.ConclusionsThis study showed a highly complex bacterial community in the apical root canal system of adequately treated teeth with persistent apical periodontitis. This suggests that this disease is characterized by multispecies bacterial communities and has a heterogeneous etiology, because the community composition largely varied from case to case.

Understanding the Maxam-Gilbert and Sanger sequencing as the first generation, in recent years there has been an explosion of newly-developed sequencing strategies, which are usually referred to as next generation sequencing (NGS) techniques. NGS techniques have high-throughputs and produce thousands or even millions of sequences at the same time. These sequences allow for the accurate identification of microbial taxa, including uncultivable organisms and those present in small numbers. In specific applications, NGS provides a complete inventory of all microbial operons and genes present or being expressed under different study conditions. NGS techniques are revolutionizing the field of microbial ecology and have recently been used to examine several food ecosystems. After a short introduction to the most common NGS systems and platforms, this review addresses how NGS techniques have been employed in the study of food microbiota and food fermentations, and discusses their limits and perspectives. The most important findings are reviewed, including those made in the study of the microbiota of milk, fermented dairy products, and plant-, meat- and fish-derived fermented foods. The knowledge that can be gained on microbial diversity, population structure and population dynamics via the use of these technologies could be vital in improving the monitoring and manipulation of foods and fermented food products. They should also improve their safety.

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