A Gram-stain-negative strain, A60T, isolated from a water well sample in Portugal, was characterized phenotypically, genotypically and phylogenetically. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain A60T belonged to the genus Citrobacter , and recN gene phylogeny revealed one strongly supported clade encompassing strain A60T and 13 other strains from public databases, distinct from currently recognized species of the genus Citrobacter . Furthermore, multilocus sequence analysis (MLSA) based on concatenated partial fusA, leuS, pyrG and rpoB sequences confirmed the classification obtained with the recN sequence. In silico genomic comparisons, including average nucleotide identity (ANI) and the genome-to-genome distance calculator (GGDC), showed 94.6 % and 58.4 % identity to the closest relative Citrobacter freundii ATCC 8090T, respectively. The ability to metabolize different compounds further discriminated strain A60T from other species of the genus Citrobacter . The G+C content of strain A60T is 52.0 %. The results obtained support the description of a novel species within the genus Citrobacter , for which the name Citrobacter portucalensis sp. nov. is proposed, with the type strain A60T (=DSM 104542T=CECT 9236T).

To gain insights into the diversification trajectories of qnrB genes, a phylogenetic and comparative genomics analysis of these genes and their surrounding genetic sequences was performed. For this purpose, Citrobacter sp. isolates ( n = 21) and genome or plasmid sequences ( n = 56) available in public databases harboring complete or truncated qnrB genes were analyzed. Citrobacter species identification was performed by phylogenetic analysis of different genotypic markers. The clonal relatedness among isolates, the location of qnrB genes, and the genetic surroundings of qnrB genes were investigated by pulsed-field gel electrophoresis (PFGE), S1-/I-CeuI-PFGE and hybridization, and PCR mapping and sequencing, respectively. Identification of Citrobacter isolates was achieved using leuS and recN gene sequences, and isolates characterized in this study were diverse and harbored chromosomal qnrB genes. Phylogenetic analysis of all known qnrB genes revealed seven main clusters and two branches, with most of them included in two clusters. Specific platforms (comprising pspF and sapA and varying in synteny and/or identity of other genes and intergenic regions) were associated with each one of these qnrB clusters, and the reliable identification of all Citrobacter isolates revealed that each platform evolved in different recognizable ( Citrobacter freundii , C. braakii , C. werkmanii , and C. pasteurii ) and putatively new species. A high identity was observed between some of the platforms identified in the chromosome of Citrobacter spp. and in different plasmids of Enterobacteriaceae . Our data corroborate Citrobacter as the origin of qnrB and further suggest divergent evolution of closely related qnrB genes/platforms in particular Citrobacter spp., which were delineated using particular genotypic markers.

One Gram-stain-positive, non-motile, non-spore-forming, catalase-negative, and coccobacilli-shaped strain, designated c10Ua161M^T, was isolated from a urine sample from a reproductive-age healthy woman. Comparative 16S rRNA gene sequence analysis indicated that strain c10Ua161M^T belonged to the genus Lactobacillus . Phylogenetic analysis based on pheS and rpoA gene sequences strongly supported a clade encompassing strains c10Ua161M^T and eight other strains from public databases, distinct from currently recognized species of the genus Lactobacillus. In silico Average Nucleotide Identity (ANI) and Genome-to-Genome Distance Calculator (GGDC), showed 87.9 and 34.3 % identity to the closest relative Lactobacillus jensenii , respectively. The major fatty acids of strain c10Ua161M^T were C_18 : 1ω9c (65.0%), C_16 : 0 (17.8%), and summed feature 8 (10.2 %; comprising C_18 : 1ω7c, and/or C_18 : 1ω6c). The DNA G+C content of the strains is 34.2 mol%. On the basis of data presented here, strain c10Ua161M^T represents a novel species of the genus Lactobacillus , for which the name Lactobacillus mulieris sp. nov. is proposed. The type strain is c10Ua161M^T (=CECT 9755^T=DSM 108704^T).

The genus Limosilactobacillus (formerly Lactobacillus ) contains multiple species considered to be adapted to vertebrates, yet their genomic diversity has not been explored. In this study, we performed comparative genomic analysis of Limosilactobacillus (22 species; 332 genomes) isolated from different niches, further focusing on human strains (11 species; 74 genomes) and their adaptation features to specific body sites. Phylogenomic analysis of Limosilactobacillus showed misidentification of some strains deposited in public databases and existence of putative novel Limosilactobacillus species. The pangenome analysis revealed a remarkable genomic diversity (only 1.3 % of gene clusters are shared), and we did not observe a strong association of the accessory genome with different niches. The pangenome of Limosilactobacillus reuteri and Limosilactobacillus fermentum was open, suggesting that acquisition of genes is still occurring. Although most Limosilactobacillus were predicted as antibiotic susceptible (83%), acquired antibiotic-resistance genes were common in L. reuteri from food-producing animals. Genes related to lactic acid isoform production (>95 %) and putative bacteriocins (70.2%) were identified in most Limosilactobacillus strains, while prophages (55.4%) and CRISPR-Cas systems (32.0%) were less prevalent. Among strains from human sources, several metabolic pathways were predicted as conserved and completed. Their accessory genome was highly variable and did not cluster according to different human body sites, with some exceptions (urogenital Limosilactobacillus vaginalis , Limosilactobacillus portuensis , Limosilactobacillus urinaemulieris and Limosilactobacillus coleohominis or gastrointestinal Limosilactobacillus mucosae ). Moreover, we identified 12 Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologues that were significantly enriched in strains from particular body sites. We concluded that evolution of the highly diverse Limosilactobacillus is complex and not always related to niche or human body site origin.