The human laryngeal microbiome: effects of cigarette smoke and reflux

Abstract

Prolonged diffuse laryngeal inflammation from smoking and/or reflux is commonly diagnosed as chronic laryngitis and treated empirically with expensive drugs that have not proven effective. Shifts in microbiota have been associated with many inflammatory diseases, though little is known about how resident microbes may contribute to chronic laryngitis. We sought to characterize the core microbiota of disease-free human laryngeal tissue and to investigate shifts in microbial community membership associated with exposure to cigarette smoke and reflux. Using 454 pyrosequencing of the 16S rRNA gene, we compared bacterial communities of laryngeal tissue biopsies collected from 97 non-treatment-seeking volunteers based on reflux and smoking status. The core community was characterized by a highly abundant OTU within the family Comamonadaceae found in all laryngeal tissues. Smokers demonstrated less microbial diversity than nonsmokers, with differences in relative abundances of OTUs classified as Streptococcus, unclassified Comamonadaceae, Cloacibacterium, and Helicobacter. Reflux status did not affect microbial diversity nor community structure nor composition. Comparison of healthy laryngeal microbial communities to benign vocal fold disease samples revealed greater abundance of Streptococcus in benign vocal fold disease suggesting that mucosal dominance by Streptococcus may be a factor in disease etiology.

A comparison of sampling methods for examining the laryngeal microbiome

“…The communities found in these pigs were similar to those that have been described in both human false vocal fold biopsies and in lesion samples[11, 14]. Communities were comprised of phyla commonly associated with animals including Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria, and Tenericutes.…”

“…To date, there are four published studies examining laryngeal microbiota using next-generation sequencing technology[11, 13–15], and most of these studies are limited by lack of a true control group. Further, given the paucity of data, there is no consensus on sampling methodology.…”

“…In a paper examining microbial communities of benign vocal fold lesions[14], all samples were tissues removed from the vocal folds as part of the standard of care and there was no control group because of the ethical implications of removing healthy vocal fold. Jetté et al[11] studied microbial shifts associated with smoking and reflux in otherwise healthy participants by examining tissue biopsies of the false vocal folds collected under local anesthesia. Given the results of the current study, swabbing the true or false vocal folds to sample the laryngeal microbiota would be a useful tool for comparing health and disease.…”

“…Common sampling methods include mucosal biopsy[5], brush[6], and swab[7], as well as lavage[8] and fecal collection [9]. The vocal folds are mucosal tissues housed within the larynx at the junction between the respiratory and gastrointestinal tracts and there is evidence that they have a distinct immunologic role [10] and microbial community[11]. The primary function of the vocal folds is airway protection; however, their vibrational properties result in voice production, making them an essential tool of human communication.…”

“…Studying the microbiota by procuring biopsies of vocal fold tissue from live humans is not ethically feasible, as even small tissue deficits have negative effects on voice[12] and it is not currently possible to regenerate vocal fold tissue. Therefore, in the literature addressing the human laryngeal microbiome, researchers have relied on tissue sampling of mucosal biopsies adjacent to the vocal folds[11, 13] or benign vocal fold lesions[14, 15]. This limitation in sampling has impeded our ability to study the role microbes play in laryngeal health and disease.…”