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

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

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

Clinical characteristics of CHM patients and expected effect of determined mutations on the structure of REP-1 protein.

*<p>HM, hand motion; LP, light perception; NLP, no light perception.</p>**<p>Brothers carrying the same mutation in Rep-1 protein.</p>†<p>effect of mutations I553X, L550P, Y504X and P179X previously analyzed by Sergeev et al. 2009.</p

Lysosomal acidification and rate of proteolytic degradation in monocytes from CHM and control patients treated with Bafilomycin-A1 (BafA1).

<p><b>a.</b> Lysosomal acidification and rate of proteolytic degradation in monocytes from CHM and control BafA1. Intralysosomal acidification measurements were performed using <i>E. coli</i> BioParticles conjugated with a pH dependent dye (pHrodo). Treatment caused an increase in lysosomal pH as evident by a decrease in the fluorescence of BioParticles (confocal images, left panel vehicle no effect, right panel cells pre- treated with BafA1 for 30 min, decreased fluorescence). <b>b.</b> Decrease in fluorescence levels of BioParticles following the treatment with BafA1 in monocytes from control and patient CHM4 measured by flow cytometry analysis at 1, 3 and 5 hours following the feeding. <b>c.</b> Representative FACS histograms showing a shift in fluorescence intensity of the CHM and control monocytes fed with BioParticles treated with BafA1 at 1, 3 and 5 h. <b>d.</b> Decreased rate of DQ-ovalbumin degradation in CHM (n = 3) and control (n = 3) patients before and after the treatment with BafA1 measured by flow cytometry analysis at 1, 3 and 5 hours following the feeding. Data expressed as a percent of fluorescence reduction in CHM and control cells treated with BafA1, compared to the non-treated (NT) cells.</p

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

Fundus photographs of the control and CHM patients.

<p><b>a.</b> CHM patient 22 y.o. characterized by RPE depigmentation and widespread RPE disruption <b>b.</b> CHM patient 74 y.o. characterized by loss of RPE and choroid, scattered pigment in macula, faint deep choroidal vessels and severely narrowed retinal vessels and optic nerve pallor (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008402#pone-0008402-t001" target="_blank"><b>Table 1</b></a>, CHM 9 and 10 respectively). <b>c.</b> Female CHM carrier, age 50 showing patchy RPE hypopigmentation without pigment dispersion and control subject. <b>d.</b> Fundus photograph of the normal eye.</p

Effect of different mutations on the structure and levels of REP-1 mRNA and protein.

<p><b>a</b>, Effect of different nonsense mutations on the structure of REP-1 protein (Q273X, I460X, M1I and K234X). <b>b</b>, Distribution and position of the mutations in the REP-1 protein, note that 4 of 9 mutations localized in the beta sheet of the REP-1 (blue) and 7 of 9 mutations (P179X, K234X, I244X, I460X, Y504 X, L550P and I553X, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008402#pone-0008402-t001" target="_blank"><b>Table 1</b></a>) localized to domain 2 of the REP-1 protein. <b>c</b>. Levels of mRNA determined by the microarray analysis of the expression profiles from monocytes and fibroblasts from CHM and control patients. Control group, n = 5; group CHM1 includes patients with low levels of REP1 mRNA, n = 7; group CHM2 includes patients with REP-1 mRNA similar to the controls, n = 6. <b>d</b>. Expression levels of REP-1 and REP-2 in different cell types derived from CHM and control patients. Lane: 1, 10 ng of rat recombinant REP-1 or 10 ng of rat recombinant REP-2 with HisTag; cell lysates (40 µg of protein for each) 2, ARPE19; 3, human fetal RPE; 4, MO- monocytes from control; 5, MO-monocytes from patient CHM4; 6, cultured human umbilical vein endothelial cells (HUVECs); 7, primary fibroblasts from control; 8, primary fibroblasts from CHM2 patient. β-actin was used as a loading control.</p

Experimental design.

<p>Collection of monocyte fractions and culture of primary dermal fibroblasts for the evaluation of gene expression and functional differences between CHM patients and age-matched controls.</p