Transplantation-associated long-term immunosuppression promotes oral colonization by potentially opportunistic pathogens without impacting other members of the salivary bacteriome

Abstract

Solid-organ transplant recipients rely on pharmacological immunosuppression to prevent allograft rejection. The effect of such chronic immunosuppression on the microflora at mucosal surfaces is not known. We evaluated the salivary bacterial microbiome of 20 transplant recipients and 19 nonimmunosuppressed controls via 454 pyrosequencing of 16S rRNA gene amplicons. Alpha-diversity and global community structure did not differ between transplant and control subjects. However, principal coordinate analysis showed differences in community membership. Taxa more prevalent in transplant subjects included operational taxonomic units (OTUs) of potentially opportunistic Gammaproteobacteria such as Klebsiella pneumoniae, Pseudomonas fluorescens, Acinetobacter species, Vibrio species, Enterobacteriaceae species, and the genera Acinetobacter and Klebsiella. Transplant subjects also had increased proportions of Pseudomonas aeruginosa, Acinetobacter species, Enterobacteriaceae species, and Enterococcus faecalis, among other OTUs, while genera with increased proportions included Klebsiella, Acinetobacter, Staphylococcus, and Enterococcus. Furthermore, in transplant subjects, the dose of the immunosuppressant prednisone positively correlated with bacterial richness, while prednisone and mycophenolate mofetil doses positively correlated with the prevalence and proportions of transplant-associated taxa. Correlation network analysis of OTU relative abundance revealed a cluster containing potentially opportunistic pathogens as transplant associated. This cluster positively correlated with serum levels of C-reactive protein, suggesting a link between the resident flora at mucosal compartments and systemic inflammation. Network connectivity analysis revealed opportunistic pathogens as highly connected to each other and to common oral commensals, pointing to bacterial interactions that may influence colonization. This work demonstrates that immunosuppression aimed at limiting T-cell-mediated responses creates a more permissive oral environment for potentially opportunistic pathogens without affecting other members of the salivary bacteriome.

Fig 1

β-Diversity comparisons between transplant and control groups. Principal coordinate plots were created based on the Jaccard distance (A) for comparison of communities of transplant and control subjects based on their composition, or from the θ_YC distance (B) for comparison of communities of transplant and control subjects according to their structure. Differences in the spatial separation of samples belonging to each group were tested using AMOVA, which was significant for the Jaccard distance. Panels C, D, E, and F show lack of clustering of samples (Jaccard) according to diabetes status (C), serum IL-6 levels (D), serum CRP levels (E), and periodontitis status (F). Data points were separated as high, medium, and low in panels D and E according to interquartile ranges. Periodontitis status in panel F was determined according to Page and Eke (43).

Fig 2

OTUs (A) and genera (B) with different prevalence in control (white) and transplant (black) subjects. Taxa with increased prevalence in control subjects are marked with #, while taxa with increased prevalence in transplant subjects are marked with a star.

Fig 3

OTUs (A) and genera (B) with different relative abundance in control (white) and transplant (black) subjects. Taxa with increased relative abundance in control subjects are marked with #, while taxa with increased relative abundance in transplant subjects are marked with a star.

Fig 4

Network correlation analysis identified a cluster (brown) associated with transplant status and containing OTUs from potentially opportunistic pathogens. Panel A shows correlations between demographic and clinical subject characteristics and the 8 different salivary OTU clusters identified in all subjects (transplant and control). Correlations are color coded, with red indicating the highest positive correlation and green the lowest negative correlation. Coefficients and P values appear in parenthesis. Panel B shows the OTUs that comprise the brown cluster and their connections. Each node in the network represents an OTU. Edge length represents adjacency between pairs of nodes. OTUs that were identified as transplant associated in individual taxa analysis are shown in yellow. Size of each node represents OTU connectivity, measured as degree centrality (number of connections with other nodes), with the bigger nodes representing those highly connected.