Abbreviations: AAI, pairwise amino acid identity; ANI, average nucleotide identity; cAAI, pairwise amino acid identity of conserved genes. †These authors also contributed equally to this work †These authors share senior authorship. Six supplementary tables and seven supplementary figures are available with the online version of this article.

Lactobacilli are a diverse group of species that occupy diverse nutrient-rich niches associated with humans, animals, plants and food. They are used widely in biotechnology and food preservation, and are being explored as therapeutics. Exploiting lactobacilli has been complicated by metabolic diversity, unclear species identity and uncertain relationships between them and other commercially important lactic acid bacteria. The capacity for biotransformations catalysed by lactobacilli is an untapped biotechnology resource. Here we report the genome sequences of 213 Lactobacillus strains and associated genera, and their encoded genetic catalogue for modifying carbohydrates and proteins. In addition, we describe broad and diverse presence of novel CRISPR-Cas immune systems in lactobacilli that may be exploited for genome editing. We rationalize the phylogenomic distribution of host interaction factors and bacteriocins that affect their natural and industrial environments, and mechanisms to withstand stress during technological processes. We present a robust phylogenomic framework of existing species and for classifying new species.

Lactobacillus species are widely used as probiotics and starter cultures for a variety of foods, supported by a long history of safe usage. Although more than 35 species meet the European Food Safety Authority (EFSA) criteria for qualified presumption of safety status, the safety of Lactobacillus species and their carriage of antibiotic resistance (AR) genes is under continuing ad hoc review. To comprehensively update the identification of AR in the genus Lactobacillus, we determined the antibiotic susceptibility patterns of 182 Lactobacillus type strains and compared these phenotypes to their genotypes based on genome-wide annotations of AR genes. Resistances to trimethoprim, vancomycin, and kanamycin were the most common phenotypes. A combination of homology-based screening and manual annotation identified genes encoding resistance to aminoglycosides (20 sequences), tetracycline (18), erythromycin (6), clindamycin (60), and chloramphenicol (42). In particular, the genes aac(3) and lsa, involved in resistance to aminoglycosides and clindamycin, respectively, were found in Lactobacillus spp. Acquired determinants predicted to code for tetracycline and erythromycin resistance were detected in Lactobacillus ingluviei, Lactobacillus amylophilus, and Lactobacillus amylotrophicus, flanked in the genome by mobile genetic elements with potential for horizontal transfer. IMPORTANCE Lactobacillus species are generally considered to be nonpathogenic and are used in a wide variety of foods and products for humans and animals. However, many of the species examined in this study have antibiotic resistance levels which exceed those recommended by the EFSA, suggesting that these cutoff values should be reexamined in light of the genetic basis for resistance discussed here. Our data provide evidence for rationally revising the regulatory guidelines for safety assessment of lactobacilli entering the food chain as starter cultures, food preservatives, or probiotics and will facilitate comprehensive genotype-based assessment of strains for safety screening.

Lactic Acid Bacteria (LAB) are a functional group of microorganisms comprising Gram-positive, catalase negative bacteria that produce lactic acid as the major metabolic end-product of carbohydrate fermentation. Among LAB, Lactobacillus is the genus including a high number of GRAS species (Generally Recognized As Safe) and many strains are among the most important bacteria in food microbiology and human nutrition, due to their contribution to fermented food production or their use as probiotics. From a taxonomic point of view, the genus Lactobacillus includes at present (October 2012), 152 validly described species, and it belongs to the family Lactobacillaceae together with genus Pediococcus, with whom it is phylogenetically intermixed. The updated phylogenetic analysis based on 16S rRNA gene sequence revealed that the family is divided into 15 groups of three or more species, 4 couples and 10 single lines of descents. In addition, other taxonomically relevant information for Lactobacillus species was collected. This study aims at updating the taxonomy of the genus Lactobacillus, presenting the phylogenetic structure of the Lactobacillaceae and discussing the clusters as possible nuclei of genera to be described in the future. It is expected that scientists and producers in the field of probiotics could benefit from information reported here about the correct identification procedures and nomenclature of beneficial strains of lactobacilli.