Phylotype-level profiling of lactobacilli in highly complex environments by means of an internal transcribed spacer-based metagenomic approach

The genus Lactobacillus is a widespread taxon, members of which are highly relevant to functional and fermented foods, while they are also commonly present in host-associated gut and vaginal microbiota. Substantial efforts have been undertaken to disclose the genetic repertoire of all members of the genus Lactobacillus, and yet their species-level profiling in complex matrices is still undeveloped due to the poor phylotype resolution of profiling approaches based on the 16S rRNA gene. To overcome this limitation, an internal transcribed spacer (ITS)-based profiling method was developed to accurately profile lactobacilli at the species level. This approach encompasses a genus-specific primer pair combined with a database of ITS sequences retrieved from all available Lactobacillus genomes and a script for the QIIME software suite that performs all required steps to reconstruct a species-level profile. This methodology was applied to several environments, i.e., human gut and vagina and the ceca of free-range chickens, as well as whey and fresh cheese. Interestingly, the data collected confirmed a relevant role of lactobacilli present in functional and fermented foods in defining the population harbored by the human gut, while, unsurprisingly perhaps, the ceca of free-range chickens were observed to be dominated by lactobacilli characterized in birds living in natural environments. Moreover, vaginal swabs confirmed the existence of previously hypothesized community state types, while analysis of whey and fresh cheese revealed a dominant presence of single Lactobacillus species used as starters for cheese production. Furthermore, application of this ITS profiling method to a mock Lactobacillus community allowed a minimal resolution level of μ0.006 ng/μl.