Characterization and comparison of bacterial communities in benign vocal fold lesions

Abstract

Background: Benign vocal fold lesions, including cysts, nodules, polyps, and Reinke's edema, are common causes of hoarseness and subsequent voice disorders. Given the prevalence of these lesions, disease etiology and pathophysiology remain unclear and their microbiota has not been studied to date secondary to the paucity of available biopsies for investigation. We sought to characterize and compare the bacterial communities in biopsies of cysts, nodules, polyps, and Reinke's edema collected from patients in Germany and Wisconsin. These samples were then compared to the communities found in healthy saliva and throat samples from the Human Microbiome Project (HMP).

Results: 454 pyrosequencing of the V3-V5 regions of the 16S rRNA gene revealed five phyla that explained most of the bacterial diversity, including Firmicutes (73.8%), Proteobacteria (12.7%), Bacteroidetes (9.2%), Actinobacteria (2.1%), and Fusobacteria (1.9%). Every lesion sample, regardless of diagnosis, had operational taxonomic units (OTUs) identified as Streptococcus, with a mean abundance of 68.7%. Most of the lesions, 31 out of 44, were indistinguishable in a principal coordinates analysis (PCoA) due to dominance by OTUs phylogenetically similar to Streptococcus pseudopneumoniae. Thirteen lesions not dominated by S. pseudopneumoniae were more similar to HMP throat and saliva samples, though 12 of them contained Pseudomonas, which was not present in any of the HMP samples. Community structure and abundance could not be correlated with lesion diagnosis or any other documented patient factor, including age, sex, or country of origin.

Conclusions: Dominance by S. pseudopneumoniae could be a factor in disease etiology, as could the presence of Pseudomonas in some samples. Likewise, decreased diversity, as compared to healthy saliva and throat samples, may be associated with disease, similar to disease models in other mucosal sites.

Keywords: Benign lesions; Larynx; Microbiota; Streptococcus; Voice disorders.

Figure 1

Taxonomic composition in benign vocal fold lesions. Mean relative abundance of the ten most common and abundant bacterial classes in cysts, nodules, polyps, and Reinke’s edema. Error bars represent standard error.

Figure 2

Comparison of bacterial community structure in benign vocal fold lesions. PCoA based on Theta Yue Clayton distances illustrating the clustering of most lesion samples, regardless of lesion diagnosis. Lesion samples are shown as circles, cysts as orange circles, nodules as yellow circles, polyps as red circles, and Reinke’s edema as blue circles.

Figure 3

Comparison of bacterial community structure in benign vocal fold lesions and throat and saliva samples from the HMP. PCoA based on Theta Yue Clayton distances illustrating the clustering of most lesion samples, regardless of lesion diagnosis with the inclusion of 15 randomly selected throat and saliva HMP samples. Lesion samples are shown as circles, cysts as orange circles, nodules as yellow circles, polyps as red circles, and Reinke’s edema as blue circles. HMP samples are shown as squares, saliva as green squares, and throat as purple squares.

Figure 4

Phylogenetic relationship and abundance of the top 100 unique Streptococcus OTUs. The top 100 unique Streptococcus OTUs along with 21 reference Streptococcus sequences were phylogenetically analyzed. Three major clades were found. Sequences from clade I dominated the lesion samples while being completely absent from HMP samples. Panel inset represents the mean relative abundance of all Streptococcus OTUs in cysts, nodules, polyps, Reinke’s edema, saliva, and throat, in addition to the portion represented by clades I, II, and III. Error bars represent standard error.