This has been shown not to be the case, as we show here there is a very little overlap between caecum and vaginal microbiota. To our best knowledge this is the first time that the BALB/c mouse vaginal bacterial community has been investigated with 454 Pyrosequencing for a full community study. This promises to be useful in futures studies of the “inheritance” of bacterial microbiome from mother to pup or vaginal microbiome related diseases such as vaginosis [28, 30]. We faced two main obstacles: The low DNA concentration in all samples, except for the caecal material and unspecific

primer binding in the tissue samples. To overcome the low DNA concentration we increased the PCR cycle number. The large cycle number essentially could amplify any kind of contamination or primer bias such as chimeras, but we adjusted buy PRI-724 the rounds of cycles to the crucial experimental negative controls as described in the material and methods. Our results are confirmed by the observed community profile of previous human lung observations (discussed in detail below) and the low abundance of this website chimera (<3% of quality trimmed sequences) [31, 32]. The second obstacle was the non-specific binding of the primers in the lung tissue sample caused by the low amounts of

SRT1720 order bacterial DNA and large amounts of eukaryotic nucleic acids. Since the risk of contamination barely left space for adjustments, we chose to do a nested PCR and amplified a ∼ 1500 bp long fragment of the 16S rRNA gene prior amplifying the hyper variable region V3/V4. Although both primer sets are universal and theoretically target all bacteria and archaea, the tendency to favor certain taxonomic groups cannot be excluded, thus one primer set should be preferred to compare the different samples. Therefor we were expecting a significantly different clustering

in beta-diversity of the lung tissue community in comparison to the BAL fluids. However the PFKL differences were small supporting our methodology. The lung has a distinct bacterial community It is not known from where we obtain our putative bacterial lung microbiota however it is most likely to be in a flux state with the environment. There is support for this notion in the hygiene hypothesis of the development of asthma and allergies [33]. We hypothesize that mice obtain the bacteria from their local environment and littermates influenced by handling by human, feed and water. But it is also a possibility that the core lung microbiome is established in utero, during and after birth in the very early life, as it is suggested with gut microbiota from human and animal studies [34–36].