Phylotype-Level Profiling of Lactobacilli in Highly Complex Environments by Means of an Internal Transcribed Spacer-Based Metagenomic Approach

Abstract

The genus 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, 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 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 species used as starters for cheese production. Furthermore, application of this ITS profiling method to a mock community allowed a minimal resolution level of<0.006 ng/μl. The genus is a large and ubiquitous taxon of high scientific and commercial relevance. Despite the fact that the genetic repertoire of 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 internal transcribed spacer to perform an accurate classification in a target population of lactobacilli with a<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. The 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.

Catching a glimpse of the bacterial gut community of companion animals: a canine and feline perspective

“…Furthermore, 16S rRNA gene‐based microbial profiling generates bacterial taxonomic composition data typically down to the genus level only, while it commonly fails in detecting underrepresented bacterial taxa, thereby causing biases in the interpretation and comparison of data from different studies (Chakravorty et al ., 2007). More recently, the so‐called internal transcribed spacer (ITS) microbial profiling methods were introduced as a tool to offer a more refined taxonomic view of the gut microbiota (Milani et al ., 2014, 2018). The ITS region is located between the 16S rRNA and 23S rRNA genes within the rRNA locus.…”

Preventing dysbiosis of the neonatal mouse intestinal microbiome protects against late-onset sepsis

“…Because standard 16S rRNA analysis poorly resolves species-level identification of the Lactobacillus genus 26 , we sought alternative approaches to identify the LAB strain(s) responsible for protection in vanc pups. First, we performed oligotyping analysis on all 16S rRNA gene reads assigned to the Lactobacillales order ( Fig.…”

Untangling Species-Level Composition of Complex Bacterial Communities through a Novel Metagenomic Approach

“…To overcome the limitations of 16S rRNA gene microbial profiling and shotgun metagenomics, we present here a pipeline for reliable and cost-effective profiling of microbial communities at the (sub)species level. In detail, we extended the internal transcribed spacer (ITS)-profiling approach described previously for bifidobacteria and lactobacilli ( 10 – 12 ) to all members of the domain Bacteria . ITS sequences are characterized by higher variability than 16S rRNA genes, thus allowing (sub)species taxonomic resolution when employed for metagenomic profiling purposes ( 10 – 12 ).…”

“…In detail, we extended the internal transcribed spacer (ITS)-profiling approach described previously for bifidobacteria and lactobacilli ( 10 – 12 ) to all members of the domain Bacteria . ITS sequences are characterized by higher variability than 16S rRNA genes, thus allowing (sub)species taxonomic resolution when employed for metagenomic profiling purposes ( 10 – 12 ). Remarkably, the ITS profiling method combines the lower cost and high sensitivity of a marker gene amplification approach with the resolution of shotgun metagenomics ( 7 , 8 ).…”