Discusión y conclusiones

To our knowledge, the work completed in this pilot study is the first evidence of using human oral plaque samples directly transferred to germ-free animals. We hypothesized that the oral microbiota from periodontally healthy and periodontitis subjects would also be reproduced in germ-free mice and aimed to generate an ‘oral microbiota humanized murine model’. The preliminary results from this work suggest the generation of a predictable model might not be obtained with the current methodology, however the results do suggest transplanted human microbes are capable of colonizing the murine oral cavity.

Using 16s deep sequencing and ProbeSEQ (citation) to characterize the microbial profiles of our samples, HOMD OTUs were detected in all analyzed samples at all time-points. Healthy and periodontitis donor samples resembled current knowledge of microbial composition and diversity [15-17]. The periodontitis donor sample harbored a community with increased microbial diversity compared to the healthy donor sample. Interestingly, this increased complexity at baseline was not conferred to the animal and potentially interfered with the traditional succession of microbial colonization[18]. The result was a final community dominated by Streptococcus sp. and limited microbial diversity. In contrast, the healthy transplanted animals had significantly more OTUs present in their community at day 42. The lack of initial microbial competition in these animals may have contributed to the community development seen throughout the duration of this study. Additionally, the healthy transplanted animals exhibited a strong trend to share increased numbers of OTUs with their baseline. Although this increased similarity was not statistically significant, the minimal sample size likely

contributed to this finding. Increasing the numbers of animals transplanted would potentially gain the necessary power to detect this suspected difference between groups.

Four ligature samples that passed quality control all showed an increase in sample complexity compared to their periodontitis counterparts. The marked inclusion of Fusobacterium sp., a known intermediary species in microbial succession[19], suggests complex communities are capable of forming from transplantation. The anatomical location of the ligature may be a more ideal location to capture the true “periodontal microbiome” adjacent to the teeth rather than the oral swab technique employed in this study.

Numerous limitations exist in this work, indicative of the nature of a pilot study. First, the animals were moved from the germ-free isolator prior to sequencing and were not returned to the isolator after transplantation due to the cost of housing and complexity of running the study in this format. As was seen in pre-transplanted samples, this likely resulted in some contamination prior to transplantation and led to the minimization of interpreting our first periodontitis transplant cohort. Additionally, the influx of new OTUs not found in our donor samples were detected at all time-points after transplantation. Interestingly, the animals transplanted with a healthy microbial donor had significantly more new OTUs in their samples. This observation may be attributed to the robust colonization that occurred in this group due to the potentially reduced colonization competition as proposed earlier. Second, the techniques employed in sample collection and subsequent sample preparation for sequencing resulted in many samples failing to meet quality control metrics. This was likely due to the minimal biomass available using the swab technique for oral microbial collection. Additionally, ligatures were left for 10 days and were occasionally missing upon retrieval. Recently, another group has discussed these issues and implemented a standardized protocol to alleviate some of these concerns[20]. Lastly,

our group recognizes a single transplantation step is likely an unrealistic approach to generate a robust model. A protocol with multiple transfections to mimic the microbial colonization patterns may lead to a profile closer resembling our current understanding of health and disease characteristics in the periodontal microbiome. However, the use of a human donor does complicate the implementation of multiple transfections as this would bring in issues with patient compliance and patient microbial recolonization following the original sampling.

In conclusion, the work outlined in this pilot study does provide evidence that microbes from human plaque samples transplanted into germ-free mice can colonize and survive for extended periods of time following transplantation. However, additional work is needed to create a reproducible ‘oral microbiota humanized murine model’ that is attractive for future studies more closely examining the host-microbial interactions that occur in periodontitis.

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