This is the first study on the composition of gut microbiota and the colonization process in infants who have experienced limited medical or dietary interventions. We also limited our analysis to the first four months of life to avoid the period of weaning, since a compositional change in gut microbiota occurs in this period (45
). Since medical interventions may be associated with increased risk of immune-related diseases in childhood (30
), studies describing a healthy gut microbiota needs to be based on children who have not been subject to such interventions. This study provides novel information of the undisturbed colonization process.
The 22 probes used in this analysis cover the most common groups identified by Palmer and coworker with several thousand probes and about 50% of their total (16
). The broad groupings used in the present paper allow for study of change in composition over time that is difficult to do when thousands of probes are used to characterize the biota.
No previous studies are directly comparable to our study, since they have all included infants exposed to at least one or more of the gut modifying factors already mentioned. The different methodology used in the studies further complicates comparison. An extensive mapping of infant gut microbiota, has previously been conducted in fourteen infants by Palmer and coworker (16
). Palmer's study included infants who had been delivered by cesarean section, had been given antibiotics, had stayed at the intensive care unit, and/or were not breastfed (information on antibiotics to the mother not given in the paper). In fact only four out of the fourteen infants in the Palmer study had not been subject to one or more such factors (16
), therefore most of these infants probably did not exhibit an unaffected colonization process. To illustrate this, Bifidobacterium
has been reported as the dominant member of the gut in healthy breastfed infants (47
), which is in accordance with our observations, but they were only minor constituents of infant microbiota in Palmer's study. Inefficient cell lysis of Gram positive bacteria which may reduce availability of chromosomal DNA, has been offered as an explanation for low detection of Bifidobacterium
in studies identifying microbes based on DNA. However, other studies based on DNA, apart from ours, have also reported a high prevalence of Bifidobacterium
). Lack of breastfeeding, antibiotics, preterm birth, and caesarean delivery are all factors which have been associated with a lack of or reduction in Bifidobacterium
species in infants (22
). The relative lack of Bifidobacterium
in some of the recent studies may therefore instead reflect differential exposure to gut microbiota modifying factors.
has been an ubiquitous inhabitant in the gut of new born infants according to older classical studies, performed in the 1970s and 80s (46
), however no longer detected in an increasing proportion of Swedish infants (56
). In contrast, in our study Enterobacteriaceae 1
(which mainly covers E.coli
was detected in seventy percent of our infants. The reported decline in E.coli
in the microbiota of Swedish infants may not be a universal trend but may also reflect exposure to gut modifying factors in subpopulations of the infants studied.
Interestingly, several clusters of infants with quite different composition of early gut microbiota were observed in our study ( & ). The factors involved in shaping these distinct sub clusters are unknown and will be further studied by our research group. However, such heterogeneity even within this subset of infants selected for “natural” early life experience highlights the large variation in infant gut composition and the need to study larger samples when aiming at describing gut composition and the importance of taking into account the many factors involved in shaping gut microbiota. Interestingly, we could not observe that the distinct clusters formed at day 4 evolved differently from each other over time. Day-to-day variation in microbial content may add random variation, making it difficult to detect such patterns. However, such random variation should not reduce the validity of our group-level findings and the trend patterns support this ().
Overall, few longitudinal studies on the evolvement of the colonization process exists and almost all are limited to cultured or species known beforehand to be constituents of the gut. Previous longitudinal studies on infant gut microbiota not restricted to already known species have been small (15
) . Our study encompassed 85 infants and the large sample size enhances the chance of identifying yet unknown constituents of microbiota and to establish both the prevalence of novel microbial constituents and their development over time with higher accuracy. More than 60% of our infants harbored microbes picked up by the Uncultured human fecal bacterium
probe which detected yet undefined non-cultivable Bacteroides
species (as well as B. dorei og B.vulgatus
). Also frequent were signaling to the different Lachnospiracea
probes. Forty-five percent of the 85 newborns had signal to the probe Lachnospiraceae 2
, which detects species belonging to Ruminococcus.
This finding is in accordance with the other studies based on DNA methodology, including cross sectional studies, (15
), where species belonging to Ruminococcus
were proposed to be a common novel constitute (15
). Noteworthy, the proportion of infants who harbored species covered by the Lachnospiracea
2 probe, showed one of the most significant increases over time (). Moreover, it was the microbial group at day 120 which seemed most influenced by early concentration of other microbes (, column 12), indicating that it could be susceptible to alterations in early gut microbiota. However, a number of other significant associations between early and later microbiota was also observed (), indicating that the presence or absence of specific microbes at an early age may be important for subsequent composition of microbiota.
Molecular methods are appropriate for studying changes over time in concentration of a given microbial group and enables elucidation of the colonization process, which is the main focus of this study. Furthermore, by assuming that a null signal is an absence of the microbe in a given sample, we have precise data on prevalence of each microbial group over time.
A limitation in all data based on fecal samples is the inability to differentiate between colonizing and transient bacteria. Also, the composition of the colonizing microbiota differs among different segments of the gastrointestinal tract (60
). However, culture based studies comparing fecal and intestinal mucosal samples have shown that a strong association exists between the two (62
), and an even stronger association could be expected when using molecular methods since microbial DNA is preserved during the passage through the intestium, supporting the use of fecal content to be used as an approximate of gut microbiota.
The present study describes the early colonization of gut bacteria in infants who have not experienced major dietary or medical interventions. It confirms the dominant role of Bifidobacterium and Enterobacteriacea reported in the older classical studies. The relative lack of these microbes in more recent studies in unselected infants could be due to the increase in medical interventions such as cesarean section.
Undefined non-cultivable species belonging to Bacteroides, as well as microbes identified as Lachnospiracea, were commonly found. Though we could not observe any cluster specific evolvement over time, strong associations were observed between some specific constituents of microbiota at day 4 and the concentration of specific microbial groups at day 120. Thus, early gut microbiota may influence later microbiota. Knowledge of the composition of a normal healthy gut microbiota and understanding the community dynamics that take place during infancy is a prerequisite for understanding the role of gut microbiota in disease and more studies among infants who have not been subject to medical interventions are needed in order to provide such knowledge.