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

Author:

Milani Christian1,Duranti Sabrina1,Mangifesta Marta1,Lugli Gabriele Andrea1,Turroni Francesca12,Mancabelli Leonardo1,Viappiani Alice3,Anzalone Rosaria1,Alessandri Giulia1,Ossiprandi Maria Cristina4,van Sinderen Douwe5,Ventura Marco12

Affiliation:

1. Laboratory of Probiogenomics, Department of Life Sciences, University of Parma, Parma, Italy

2. Microbiome Research Hub, University of Parma, Parma, Italy

3. GenProbio Srl, Parma, Italy

4. Department of Veterinary Medical Science, University of Parma, Parma, Italy

5. APC Microbiome Ireland and School of Microbiology, University College Cork, Cork, Ireland

Abstract

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 , 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 <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 Lactobacillus 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.

Funder

Science Foundation Ireland

Ministero dell'Istruzione, dell'Università e della Ricerca

Publisher

American Society for Microbiology

Subject

Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology

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