Identification and Characterization of Human Breast Milk and Infant Fecal Cultivable Lactobacilli Isolated in Bulgaria: A Pilot Study

During the last few decades, the main focus of numerous studies has been on the human breast milk microbiota and its influence on the infant intestinal microbiota and overall health. The presence of lactic acid bacteria in breast milk affects both the quantitative and qualitative composition of the infant gut microbiota. The aim of this study was to assess the most frequently detected cultivable rod-shaped lactobacilli, specific for breast milk of healthy Bulgarian women and fecal samples of their infants over the first month of life, in 14 mother-infant tandem pairs. Additionally, we evaluated the strain diversity among the most common isolated species. A total of 68 Gram-positive and catalase-negative strains were subjected to identification using the MALDI-TOF technique. Predominant cultivable populations belonging to the rod-shaped lactic acid bacteria have been identified as <i>Lacticaseibacillus rhamnosus</i>, <i>Limosilactobacillus fermentum</i>, <i>Lacticaseibacillus paracasei</i>, and <i>Limosilactobacillus reuteri</i>. Also, we confirmed the presence of <i>Lactiplantibacillus plantarum</i> and <i>Lactobacillus gasseri.</i> Up to 26 isolates were selected as representatives and analyzed by 16S rRNA sequencing for strain identity confirmation and a phylogenetic tree based on 16S rRNA gene sequence was constructed. Comparative analysis by four RAPD primers revealed genetic differences between newly isolated predominant <i>L. rhamnosus</i> strains. This pilot study provides data for the current first report concerning the investigation of the characteristic cultivable lactobacilli isolated from human breast milk and infant feces in Bulgaria.