A Characterization of the Oral Microbiome in Allogeneic Stem Cell Transplant Patients

<div><h3>Background</h3><p>The mouth is a complex biological structure inhabited by diverse bacterial communities. The purpose of this study is to describe the effects of allogeneic stem cell transplantation on the oral microbiota and to examine differences among those patients who acquired respiratory complications after transplantation.</p> <h3>Methodology/Principal Findings</h3><p>All patients were consented at the National Institutes of Health, Clinical Center. Bacterial DNA was analyzed from patients' oral specimens using the Human Oral Microbe Identification Microarray. The specimens were collected from four oral sites in 45 allogeneic transplantation patients. Specimens were collected at baseline prior to transplantation, after transplantation at the nadir of the neutrophil count and after myeloid engraftment. If respiratory signs and symptoms developed, additional specimens were obtained. Patients were followed for 100 days post transplantation. Eleven patients' specimens were subjected to further statistical analysis. Many common bacterial genera, such as <em>Streptococcus, Veillonella, Gemella</em>, <em>Granulicatella</em> and <em>Camplyobacter</em> were identified as being present before and after transplantation. Five of 11 patients developed respiratory complications following transplantation and there was preliminary evidence that the oral microbiome changed in their oral specimens. Cluster analysis and principal component analysis revealed this change in the oral microbiota.</p> <h3>Conclusions/Significance</h3><p>After allogeneic transplantation, the oral bacterial community's response to a new immune system was not apparent and many of the most common core oral taxa remained unaffected. However, the oral microbiome was affected in patients who developed respiratory signs and symptoms after transplantation. The association related to the change in the oral microbiota and respiratory complications after transplantation will be validated by future studies using high throughput molecular methods.</p> </div

Proportion Present of Common Bacterial Taxa.

<p>Eighteen of the most prevalent bacterial probes in the entire sample and their mean proportions are shown. Panel A displays before (red) or after (blue) transplantation and panel B shows patients with NoRSS (orange) or with RSS (purple). The proportion present of each probe and standard error bars are displayed in order according to the mean proportion present (Each error bar is constructed using 1 standard error from the mean.). Each of the eighteen positive probes mean proportions were calculated for all specimens before transplant (n = 39), after transplant (n = 74) or with NoRSS (n = 61) and with RSS (n = 52). NoRSS = patients who do not develop respiratory signs and symptoms. RSS = patients who develop respiratory signs and symptoms. Streptococcus Cluster III includes all Streptococcus species; Veillonella Cluster II includes: <i>V.atypica, V.parvula, V.dispar</i>, BU083; Streptococcus Cluster IV includes: <i>S.anginosus, S.intermedius</i>, 17 bases match <i>S. sinensis, S.pneumoniae, S.parasanguis, S.oralis, S.mitis, S.infantis</i>; Veillonella Cluster IV includes: <i>V.parvula</i>, BU083, <i>V.dispar</i>; Streptococcus Cluster II includes: <i>S.sanguinis, S.salivarius</i>, strain H6.</p

Genera Factor Loading of Principal Component Analysis.

<p>Factor loading plot of proportion of positive probes further illustrates the PCA plot from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047628#pone-0047628-g004" target="_blank">Figure 4</a>. The loading of the 43 genera show how each genus contributed to the score plot.</p

Principal Component Analysis by Genera.

<p>Patients with NoRSS are represented by red circles and those with RSS are represented with blue triangles. First principal component score (PC 1) vs. second principal component score (PC 2) plots the proportion of positive probes. Plot displays the first two principal components that represent 42.2% of the variability in the data matrix. NoRSS = patients who do not develop respiratory signs and symptoms. RSS = patients who develop respiratory signs and symptoms.</p

Sampling information.

<p>Sample descriptions including time and site obtained for each patient and totals. Specimens obtained from plaque (P), saliva (S), buccal brushings (B) and tongue brushings (T). Baseline specimens obtained at start of study. Nadir of absolute neutrophil count (ANC) is after transplant when ANC is at its lowest point. Engraftment is after transplant when ANC remained at 0.5×109/liter for two days. RSS represents patients that developed respiratory signs and symptoms after transplant and NoRSS represents patients who did not develop this complication.</p

Two Way Cluster of Genera by Patient.

<p>Fraction of positive probes all sites and all times by genera and patient. The two way hierarchical cluster analysis using Ward's distance measure without standardization was calculated. Each probe was converted to a 1 (present) or a 0 (absent) based on the intensity range of the microarray data. The 155 probes were then divided among 43 genera and the proportion of positive probes within each genus was calculated. Cluster A consists of mostly NoRSS and Cluster B consists of RSS patients with one exception (017). Frequently occurring probes are in Cluster D while less frequently occurring probes are Cluster C. Supplementary material (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047628#pone.0047628.s003" target="_blank">Table S1</a>) lists the probes and the assigned genera for this figure. NoRSS = patients who do not develop respiratory signs and symptoms. RSS = patients who develop respiratory signs and symptoms. [Note: Two probes (<i>Sphaerocytophaga</i> species strain S3) are listed in <i>Sphaerocytophaga</i> genus but are considered as synonymous with <i>Capnocytophaga</i> genera in the Human Oral Microbiome Database (HOMD) 15]. <i>Selenomonas</i> species Oral Clone CS002 oral taxon 131 remains in <i>Selenomonas</i> genus despite being re-classified into <i>Mitsuokella</i> genera Oral Clone CS002 by HOMD. <i>Eubacterium</i> PUS9170_MCE10174 remains in the <i>Eubacterium</i> genus.]</p

Comparisons of Principal Component Analysis Plots: Before vs. After Transplantation and NoRSS vs. RSS.

<p>Bivariate plots of principal component 1 (PC1) and principal component 2 (PC2) compare before vs. after transplantation in Plot A and RSS vs. NoRSS in Plot B. Plot A shows overlapping density ellipse between before transplantation (blue cross) and after transplantation (red open circle) groups may indicate lack of significant difference in expression between the two groups. Plot B depicts a density ellipse drawn with 50% confidence of the mean for each group. Lack of overlap of density ellipse between the RSS (blue cross) and NoRSS (red open circle) samples indicates a possible significant difference in expression between the two groups. NoRSS = patients who do not develop respiratory signs and symptoms. RSS = patients who develop respiratory signs and symptoms.</p

Percent of positive probes per phyla.

<p>In the analysis of 11 patients there were 155 probes divided among 8 different phyla. This chart represents the percentage of probes per phyla ordered by most common to less common.</p

Bacterial Taxa by Site; Before and After Transplantation.

<p>Fraction of positive bacterial probes for each patient, before and after transplantation for saliva (A), plaque (B), buccal (C) and tongue (D) brushings. Each point represents a fraction of positive probes for each patient. Box plots show medians and lower and upper quartiles. The sample size varies slightly by site and by time for each site does not include all eleven patients.</p

Cluster Analysis of RSS vs. NoRSS.

<p>Clinical specimens (113) from 11 patients (rows) by probes (73) (column). To be included each probe must have a median sum expression of 17 positive specimens for that probe. The sum was taken across the 113 specimens for each bacteria probe and the median was determined. A total of 73 out of 155 probes passed this requirement and were used in the two-way cluster (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047628#pone.0047628.s004" target="_blank">Table S2</a> lists the probes). The specimens were first divided into four groups RSS before and after transplant vs. NoRSS before and after transplant groups. Clustering was performed in each of the four groups. A second clustering was applied to probes. Cluster A specimens are composed of probes that were positive for <i>Camplylobacter rectus</i> and/or <i>C. concisus</i>. Cluster B is composed of positive probes <i>Campylobacter rectus</i> and/or <i>C. concisus,</i> two probes for <i>Streptococcus,</i> namely <i>Streptococcus intermedius</i> and/or <i>S. constellatus</i> and <i>Streptococcus anginosus</i> and/or <i>S.intermedius</i>. Also included in Cluster B are positive probes for <i>Dialster invisus, Eubacterium infirmum</i> and five different probes for the genus <i>Selenomonas</i>. Cluster C is composed of positive probes for <i>Parvimonas micros Dialaster invisus</i> and <i>Eubacterium saburreum</i>. Cluster D and E represent common oral bacterial taxa. Analysis was performed using JMP (Cary, NC), hierarchical two-way clustering with Ward's distance measure applied to the normalized fluorescence intensity signal from the microarray data, on a 0 to 5 scale. NoRSS = patients who do not develop respiratory signs and symptoms. RSS = patients who develop respiratory signs and symptoms.</p