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

Abstract

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, 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 ITS-based profiling method was developed to accurately profile lactobacilli at 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, cecum of free range chickens, as well as whey and fresh cheese. Interestingly, 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 cecum of free range chickens was 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 <0.006 ng/μl. Importance: The genus Lactobacillus is a large and ubiquitous taxon of high scientific and commercial relevance. Despite the fact that the genetic repertoire of lactobacilli species has been extensively characterized, the ecology of this genus has been explored by metataxonomic techniques that are accurate down to the genus or phylogenetic group level only. Thus, the distribution of lactobacilli in environmental or processed food samples is relatively unexplored. The profiling protocol described here relies on the use of the Internally Transcribed Spacer to perform an accurate classification in a target population of lactobacilli with <0.006 ng/μl sensitivity. This approach was used to analyze five sample types collected from both human and animal host-associated microbiota as well as from the cheese production chain. Availability of a tool for species-level profiling of lactobacilli may be highly useful for both academic research and a wide range of industrial applications.