The bacterial composition of gut microbiota in early life is of paramount relevance for the health of infants and also later on, as colonization by commensal human intestinal bacteria stimulates a range of important functions, from postnatal intestinal development to maintenance of the mucosal barrier and nutrient absorption (1). The massive use of culture-independent, DNA-based techniques has improved our knowledge about the presence of bacterial species not easily cultured. The infant core microbiota is composed of species belonging to the phylum Firmicutes (including Clostridium, Enterococcus, Lactobacillus, and Ruminococcus) or to the phylum Bacteroidetes (including Bacteroides and Prevotella genera), and these phyla constitute over 90% of the known phylogenetic categories found in the human intestine (2, 3). The use of DNA-based techniques confirmed that breast-fed infants have a microbiota dominated by Bifidobacterium (4, 5) with a lower presence of Escherichia coli (E. coli), Clostridium difficile (C. difficile), the Bacteroides fragilis (B. fragilis) group, and lactobacilli than the microbiota of exclusively formula-fed infants (6).

In recent years, members of Lachnospiraceae, a family of the order Clostridiales, have been detected, using DNA-based approaches, as constituents of mammalian intestinal microbiota, including that of humans (7). Lachnospiraceae constitutes one of the major taxonomic groups of the human gut microbiota that degrade complex polysaccharides to short-chain fatty acids, including acetate, butyrate, and propionate, that can be used for energy by the host (8). Other animals commonly harbor Lachnospiraceae, with herbivores having a higher abundance than carnivores (9). The wide range of functions carried out by Lachnospiraceae may influence their relative abundance in gut communities of different hosts.

The Lachnospiraceae family is formed by 24 named genera, including Ruminococcus, Blautia, Dorea, and Lachnoanaerobaculum as well as a number of incertae sedis strains (10) sharing a high degree of similarity among their 16S rDNA sequences (11, 12). Some members of this family are non-spore-forming, but all of them are strictly anaerobes (12). In human adults, members of this family have been associated with protection against C. difficile infections (13) and obesity (14). They are also known as potent short fatty acid producers (15). However, despite their apparent importance, little is known about their presence and possible roles played by these bacteria in the early life of humans.

Members of this family, mainly belonging to the genus Ruminococcus, have been identified in the stool samples of neonates and infants (16–18), while it was impossible to detect their presence in 46 babies delivered by natural delivery or cesarean section (19). Their presence was also confirmed in babies fed soy milk (20) or goat milk (21). Results of this latter study suggested a relevant presence of members of the species of Ruminococcus gnavus (R. gnavus) in babies fed breast milk or goat milk formula compared with babies fed cow milk formula, while Bifidobacteriaceae were abundant in the microbiota of infants in all three groups.

The present study aimed to assess the presence of Lachnospiraceae as a component of the infant microbiota in a group of 25 infants, focusing on Ruminococci and in particular the presence of R. gnavus, from the first month up to the second year of life by means of denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR). To further assess our hypothesis that Lachnospiraceae could be a dominant bacterial group in the infant gut from the first month to the second year of life, qPCR was used to quantitate the total Bifidobacterium species for a reference.

The DGGE profiles showed that the bacterial community of all samples produced different banding patterns; thus, the composition of the Lachnospiraceae family was different between subjects. Results suggested that the vertical distribution of the DGGE profiles did not depend on the age of the subject. It was influenced by interpersonal variability. The alignment showed that the most prevalent species recovered in samples were Blautia luti (B. luti), Blautia producta (B. producta), B. wexlerae, Lachnoanaerobaculum orale (L. orale), Dorea formicigenerans (D. formicigenerans), E. rectale, and R. gnavus, as reported in Table 1. The most interesting result obtained from the DGGE was that R. gnavus was present in 16 out of 25 samples (Figure 1A). The 16 positive samples for the presence of R. gnavus were from 10 of the 17 babies born by natural delivery and 6 of the 8 babies born by C-section. Furthermore, samples from 11 of the 18 breast-fed babies and 5 of 7 formula-fed babies displayed the band corresponding to R. gnavus. Combining the delivery and the feeding conditions: 4 of the 16 positive samples were C-section delivered and breast-fed, 2 were C-section delivered and formula-fed, 7 were vaginally delivered and breast-fed, and 3 were vaginally delivered and formula-fed babies. The DGGE preliminary results suggest that the presence the R. gnavus is not strictly dependent on the delivery and feeding mode, and this hypothesis is corroborated by the cluster analysis (Figure 1B).

Quantitative PCR results indicated that the mean number of 16S rRNA gene copies detected in fecal samples using primers for the Clostridium XIVa group was 9.507 ± 2.397 (log 16S rRNA gene copies per gram of wet feces), while primers specific for the Blautia genus detected 7.863 ± 8.213 log 16S rRNA gene copies per grams of wet feces. R. gnavus was present at a level of 8.722 ± 9.289 (log 16S rRNA gene copies per gram of wet feces), and this microorganism was detected in 22 of 25 (88%) samples, apparently due to the higher sensitivity of the PCR technique compared with the DGGE. The quantification of Bifidobacteria by qPCR showed that it is present in all samples at a level of 9.976 ± 10.191 (log 16S rRNA gene copies per gram of wet feces).

Lachnospiraceae is a bacterial family known to be abundant in the intestinal ecosystem, and recently, there has been a growing interest in the presence and role of these microorganisms in the adult microbiota. R. gnavus was found in the intestinal tract of 90% of adults (2) and sharply increased in pathological conditions, such as inflammatory bowel diseases (IBD) (33). It has been described that the R. gnavus ATCC 29149 strain possesses the complete Nan cluster involved in sialic acid metabolism for the production of an intramolecular trans-sialidase (34). Moreover, the analyses of metagenomes confirm that this enzymatic pathway is present in healthy subjects and is predominant in IBD metagenomes (34). It has also been recently demonstrated that R. gnavus produces iso-bile acids. The iso-bile acids detoxification pathway influences the growth of one of the predominant genera in the human gut, i.e., the Bacteroides (35). These recent studies underline the importance of understanding the biological role of the Lachnospiraceae family and, in particular of R. gnavus, in the complexity of the human microbiota in babies and adults.

However, the presence and role of Lachnospiraceae members, and in particular R. gnavus, in the infant gut is still unclear. A deeper understanding of the prevalence and diversity of bacteria in the gut of infants is important for human gut ecology and future nutrition research. In our study, the total number of subjects harboring R. gnavus, evaluated using the 16S rRNA gene, were higher compared with other reports in literature (16, 17, 19, 20) (Table S2 in Supplementary Material), supporting the need to develop specific molecular tools for this bacterial group. The difference in subjects positive for the presence of R. gnavus could be due to the different DNA extraction methods and different sets of primers used for the DGGE analysis. Several studies (16, 19, 20) found the Ruminococcus genus using the DGGE analysis with the universal primers U968-GC-f and L1401-r (36) for the amplification of the V₆–V₈ 16S rRNA region. Yu and Morrison (37) reported that this primer set allows a minor recovery of bacterial richness. Other universal primers for the 16S rRNA gene were employed to evaluate the bacterial populations harbored by the infant gut, such as HDA-1-GC and HAD-2 (18). The lower presence of Ruminococcus spp. could be attributed to the use of a set of primers targeting the V₂–V₃ region of the 16S rRNA of bacteria, not specifically for the Clostridium XVIa group like we used. Furthermore, the assessment of different techniques, such as temperature gradient gel electrophoresis, with universal primers (17) could lead to an underestimation of the Ruminococcus spp. In the present study, we failed to detect R. gnavus 16S rRNA gene sequences in 9 out of 25 stool specimens by DGGE. For only three specimens, we found no presence by DGGE or qPCR, although this could be due to the limit of detection of the molecular techniques or to the coverage of the primers used in this study. This result suggests that R. gnavus colonization in the infant intestine is variable across individuals and not strictly linked to the type of feeding or the delivery mode. As reported in a previous study (38), the presence of the Ruminococcus genus was identified by a molecular method as another additional anaerobic bacterial group that has to be considered as “dominant” after the very first days of life, together with Bifidobacteria. Our results further support this hypothesis and strongly suggest that the presence of R. gnavus is predominant in the infant gut at levels as high as Bifidobacteria and is not dependent on the type of delivery and feeding. This finding is particularly relevant because Ruminococci and Bifidobacteria share metabolic pathways involved in complex sugar degradation (39) and in the degradation of mucin (40). Thus, the presence of high levels of R. gnavus is a notable finding and supports the suggestions that the release of sugars by the mucin degradation might be important for succession by other bacteria (41). Further investigations are required to ascertain whether the high level of R. gnavus has functional consequences. Blanton et al. suggested a putative role of R. gnavus in ameliorating growth and metabolic abnormalities in animals receiving fecal transplantation from malnourished babies aged 6–18 months (42). Their results support the ability of R. gnavus in promoting protein synthesis and lean body mass formation instead of amino acid oxidation. The results obtained in a murine model indicate that R. gnavus may help preventing malnutrition and clearly support the relevant role of this organism in the assessment of infant gut microbiota. Our pilot study has several limitations that must be acknowledged. Among these, a major limitation is represented by the low number of recruited subjects, and thus, investigations on a wider cohort of babies are necessary to get a deeper insight into the microbial ecology of R. gnavus in the gut. Nevertheless, the present study represents the first work supporting using the qPCR technique to quantify the presence of R. gnavus in the intestinal tract of infants until the second year of life, and the results support the need for further efforts to verify and elucidate the biological role of this organism in the early stages of life.

VS drafted the manuscript, collected samples, and performed DNA extraction and quantitative PCR. VP jointly led the study and revised the manuscript. FM performed the denaturing gradient gel electrophoresis and the cluster analysis and revised the manuscript. EP revised the manuscript. LM conceived and designed the study and revised the manuscript. All the authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.