We previously reported that supplementation with L. reuteri DSM 17938 did not reduce crying time in breastfed and formula-fed infants with colic16. However, the L. reuteri colonization rates were lower than expected, potentially affecting the Baby Biotics trial results. In this substudy, we examined the relationship between L. reuteri colonization and microbiological outcomes (E. coli colonization and microbial diversity), intestinal inflammation, and crying time in a subset of infants from the trial. L. reuteri colonization rates (45% at ~ 4 months of age) in the current study were lower than those previously reported in other pediatric studies (92% at ~ 2 months11, and 65% at ~3 months18). The reduction in colonization rates with increasing infant age suggest age at probiotic administration and/or age at sample collection, as well as the use of different microbiological detection methods (culture-dependent versus culture-independent methods in our study), may affect L. reuteri detection11,18.We found median colonization density of L. reuteri to be 3.6 × 105 genome equivalents/ml in infants who were colonized with L. reuteri, which is in line with previous research that found mean L. reuteri colonization densities of 104–105 CFU/g of feces after supplementation11,19. Microbial diversity was similar in infants colonized or not colonized with L. reuteri, consistent with Roos et al. who found that treatment with L. reuteri did not affect the global composition of the bacterial community20. We found no difference in calprotectin levels in infants colonized or not colonized by L. reuteri, and no association between calprotectin levels and crying time at day 28. However, we previously reported that infants who had a reduction in crying time of at least 50% had lower calprotectin levels, suggesting gut inflammation may be implicated in infant colic, although the precise relationship remains unclear16. Crying time decreased in infants from day 0 to day 28 regardless of L. reuteri colonization status, in line with the natural resolution of infant colic that typically occurs when an infant reaches three to four months of age21. There were two infants in the L. reuteri colonized group with lower crying times at day 0 than were observed in the not colonized group (approximately 60 min/day, versus minimum of 150 min/day). This may have decreased the ability to detect a reduction in crying time from day 0 to day 28 in the infants colonized with L. reuteri, however we still found a reduction in median crying time. We found no difference in crying time at day 28 between infants colonized or not colonized by L. reuteri. These findings were consistent with the original Baby Biotics trial results, and in contrast to previous reports that L. reuteri treatment is effective in reducing crying time in infants with colic when crying is assessed by treatment allocation11,12,13,14,15. These divergent findings may reflect differences in geographic location of previous studies (Italy, Poland, China and Canada) and rates of exclusive breastfeeding (majority of infants in previous studies versus 35% in our study). Geography and method of feeding are known to impact on gut microbiota22,23,24,25, therefore our study emphasizes that results from other settings may not be generalizable to our study population, or to Australian infants in general. In infants colonized by L. reuteri, we observed an unexpected positive relationship between L. reuteri density and crying time. Although this finding may generate concerns that high density of L. reuteri could exacerbate symptoms of colic, there does not appear to be a link with gut inflammation as there was no association between L. reuteri density and calprotectin. It is plausible that some infants may have an underlying gut environment that is associated with higher crying and also facilitates high-density L. reuteri growth.

In our study, we found no evidence linking E. coli to gut inflammation as measured by calprotectin. We found a negative association between E. coli colonization density and microbial diversity, although this was only detected with one of the enzymes used in the diversity assay. E. coli has been associated with Crohn’s Disease, with decreases in microbial diversity compared with healthy participants26,27, suggesting a possible pathophysiological relationship between GI E. coli colonization and microbial diversity. Savino et al. found that infants who responded to L. reuteri treatment (a reduction of ≥50% crying time) had a reduction in fecal E. coli, so our finding of no difference in E. coli colonization is consistent with the outcome that treatment with L. reuteri did not reduce crying time in this study population. There was moderate evidence that infants colonized with E. coli had a smaller reduction in crying from baseline to day 28, however there was no difference between crying time at day 28 when assessed by E. coli colonization status. It has been previously reported that L. reuteri administration was associated with a decreased presence of E. coli, suggesting a potential negative interaction between these two species11. However, we did not observe a negative relationship between L. reuteri and E. coli, and in fact, 93% of infants colonized with L. reuteri also had E. coli detected. This suggests that the findings of Savino et al. may have been due to an indirect rather than direct relationship between the two species, and/or could be another example of differences attributable to geography and feeding methods.

To our knowledge, this is the first study to investigate microbiological changes in infant colic by L. reuteri colonization status. The participants in our study are representative of help-seeking parents and carers from the general population. Because Australian pediatricians do not deliver primary care, families often present to the emergency department for common childhood concerns, especially for conditions such as colic where the burden typically occurs outside business hours. Additionally, this study included both breast and formula-fed infants and did not restrict the mother’s diet to make the probiotic intervention more generalizable. This is in contrast to the majority of infant colic studies that use populations of exclusively (or predominately) breastfed infants11,12,13,14,15, and have mothers or infants on a cow’s milk-free diet11,12,14. However, this study had several limitations. Only a subset of samples from the original trial were available for the analyses conducted in this pilot project. We performed laboratory analyses at a single time point at the end of treatment when crying time had already reduced, which may have blunted the ability to detect potential differences linked to crying time. The infant microbiome is highly variable, and it is plausible that transient changes due to probiotic colonization may have occurred earlier. Additionally, while we used robust culture-independent molecular methods to identify L. reuteri and E. coli, the qPCR primers and probes may detect other closely related species, such as the closely related Shigella spp. for the E. coli assay28,29,30. Nevertheless, only infants in the probiotic group had detectable L. reuteri, suggesting our assay was specific for the probiotic strain. Lastly, parents were instructed to deliver the supplement using a spoon or dropper; it is not known whether the route of administration impacted L. reuteri colonization. Advances in molecular methods and sequencing technology have enabled more detailed characterization of the microbiome including identification of more subtle differences in the bacterial communities present. Employing one of these methods may provide further information on differences in the GI microbiota and greater insight into potential impacts of L. reuteri treatment on the gut microbiome.

We found no reduction in crying time in infants colonized with L. reuteri and identified an unexpected positive association between L. reuteri density and crying time. Our results support the finding of the original Baby Biotics trial that L. reuteri supplementation did not reduce crying time in infants with colic in Melbourne, Australia. We also found a negative association between E. coli colonization density and microbial diversity, suggesting a possible pathophysiological relationship between E. coli colonization and microbial diversity. Our findings suggest L. reuteri supplementation may not be an appropriate treatment for infants with colic in some geographic settings, and that the impact of such an intervention on intestinal microbiota composition remains unclear. As our study included a small subset of infants from the Baby Biotics randomized trial, additional studies with larger sample size are required to further investigate the role of the gut microbiome in infant colic and the utility of probiotic treatment in different populations.