The vaginal microbial community plays a vital role in maintaining women’s health. Understanding the precise bacterial composition is challenging because of the diverse and difficult-to-culture nature of many bacterial constituents, necessitating culture-independent methodology. During a natural menstrual cycle, physiological changes could have an impact on bacterial growth, colonization, and community structure. The objective of this study was to assess the stability of the vaginal microbiome of healthy Canadian women throughout a menstrual cycle by using cpn60-based microbiota analysis. Vaginal swabs from 27 naturally cycling reproductive-age women were collected weekly through a single menstrual cycle. Polymerase chain reaction (PCR) was performed to amplify the universal target region of the cpn60 gene and generate amplicons representative of the microbial community. Amplicons were pyrosequenced, assembled into operational taxonomic units, and analyzed. Samples were also assayed for total 16S rRNA gene content and Gardnerella vaginalis by quantitative PCR and screened for the presence of Mollicutes by using family and genus-specific PCR.

Background: The genus Campylobacter includes many species, some of which are known human and animal pathogens. Even though studies have repeatedly identified domestic dogs as a risk factor for human campylobacteriosis, our understanding of Campylobacter ecology in this reservoir is limited. Work to date has focused primarily on a limited number of species using culture-based methods. To expand our understanding of Campylobacter ecology in dogs, a collection of fecal samples from 70 healthy and 65 diarrheic pet dogs were examined for the presence and levels of 14 Campylobacter species using quantitative PCR. Results: It was found that 58% of healthy dogs and 97% of diarrheic dogs shed detectable levels of Campylobacter spp., with C. coli, C. concisus, C. fetus, C. gracilis, C. helveticus, C. jejuni, C. lari, C. mucosalis, C. showae, C. sputorum and C. upsaliensis levels significantly higher in the diarrheic population. Levels of individual Campylobacter species detected ranged from 10³ to 10⁸ organisms per gram of feces. In addition, many individual samples contained multiple species of Campylobacter, with healthy dogs carrying from 0-7 detectable species while diarrheic dogs carried from 0-12 detectable species. Conclusions: These findings represent the largest number of Campylobacter species specifically tested for in animals and is the first report to determine quantifiable levels of Campylobacter being shed from dogs. This study demonstrates that domestic dogs can carry a wide range of Campylobacter species naturally and that there is a notable increase in species richness detectable in the diarrheic population. With several of the detected Campylobacter species known or emerging pathogens, these results are relevant to both ecological and public health discussions.

Background: Formation of operational taxonomic units (OTU) is a common approach to data aggregation in microbial ecology studies based on amplification and sequencing of individual gene targets. The de novo assembly of OTU sequences has been recently demonstrated as an alternative to widely used clustering methods, providing robust information from experimental data alone, without any reliance on an external reference database. Results: Here we introduce mPUMA (microbial Profiling Using Metagenomic Assembly, http://mpuma.sourceforge.net), a software package for identification and analysis of protein-coding barcode sequence data. It was developed originally for Cpn 60 universal target sequences (also known as Gro EL or Hsp 60). Using an unattended process that is independent of external reference sequences, mPUMA forms OTUs by DNA sequence assembly and is capable of tracking OTU abundance. mPUMA processes microbial profiles both in terms of the direct DNA sequence as well as in the translated amino acid sequence for protein coding barcodes. By forming OTUs and calculating abundance through an assembly approach, mPUMA is capable of generating inputs for several popular microbiota analysis tools. Using SFF data from sequencing of a synthetic community of Cpn 60 sequences derived from the human vaginal microbiome, we demonstrate that mPUMA can faithfully reconstruct all expected OTU sequences and produce compositional profiles consistent with actual community structure. Conclusions: mPUMA enables analysis of microbial communities while empowering the discovery of novel organisms through OTU assembly.