## 4 Discussion
Although this observation of potential engraftment is encouraging, the authors note that we were not able to evaluate long-term engraftment in this study. Studies are mixed as to whether orally administered probiotics can stably or transiently engraft [41; 42]; while stable engraftment appears possible for some probiotics, this may be dependent on the gut microbiome of the host as well as other host-related factors [41; 43; 44]. This was demonstrated by a study of _Bifidobacterium longum_ AH1206 which reported long-term persistence (> 6 months) in only 30% of study participants [41], as well as mouse studies that suggest the presence of an exclusive metabolic niche enablesengraftment in the gut microbiota [43]. It has been suggested that prebiotics improve engraftment [45]; therefore, it would be interesting to test for engraftment of the MSB strains with and without prebiotics and to determine whether any specific prebiotics facilitate improved engraftment of these strains. MegaSporeBiotic(tm) supplementation has been shown to result in changes in community activity and composition, such as increased propionate levels, decreased lactate levels, and increased _Akkermansia muciniphila_, _Bifidobacteria_ spp., and Firmicutes in an M-SHIME(r) model using the fecal microbiota of adult donors under healthy conditions [21]. Because of this, we hypothesized that MegaSporeBiotic(tm) treatment may enhance recovery following antibiotic-induced dysbiosis in an M-SHIME(r) model using fecal microbiota of baby donors. While the chambers treated with MegaSporeBiotic(tm) did recover both microbial community activity and composition following antibiotic-induced dysbiosis, the recovery was not enhanced relative to untreated (i.e., spontaneous recovery). A study in premature infants found that supplementing probiotics containing _B. longum_, _Lactobacillus acidophilus_, and _Enterococcus faecalis_ within two days of receiving cefotaxime antibiotics resulted in increased beneficial bacteria relative to the infants who received antibiotics only [46], indicating that there may be a role for probiotics in antibiotic-induced dysbiosis recovery. Another study evaluated probiotic supplementation (_Bifidobacterium breve_, _Propionibacterium freundenreichii_, _Lactobacillus rhamnosus_ LC705, and _L. rhamnosus_ GG) to mothers during pregnancy and their infants after birth for 3 months [40]. Using fecal samples collected from the infants at 3 months, the study reported that there was a strong effect of antibiotic use and birth mode (i.e., vaginal birth versus caesarean section) in the group that did not receive probiotics, which was eliminated in the group that did, indicating that consistent probiotic use could mitigate the damaging effects of antibiotic use on the fecal microbiota. Together, these studies may indicate the importance of earlier probiotic treatment (i.e., within two days of antibiotic treatment or concurrently with antibiotic treatment) for an improved effect of probiotics in babies treated with antibiotics. Our study had several limitations. This was an in vitro study. As such, the results cannot directly translate to biological responses, meaning that studies in vivo are needed to confirm these findings. Probiotic treatment was initiated following five days of antibiotic treatment; thus, we were not able to evaluate the potential benefits of earlier or simultaneous probiotic supplementation. Additionally, the study was not designed to evaluate whether the probiotic strains established themselves within the gut microbiota; therefore, it is unclear whether the lack of effect was due to a lack of transient persistence or a true lack of effect. Finally, in the real world, antibiotics are given to treat an infection; however, in our study, the donors were healthy and did not have a known infection.