Proper preservation of stool samples to minimize microbial community shifts and inactivate infectious agents is important for self-collected specimens requiring shipment to laboratories when cold chain transport is not feasible. In this study, we evaluated the performance of six preservation solutions (Norgen, OMNI, RNAlater, CURNA, HEMA, and Shield) for these aspects. Following storage of human stool samples with these preservatives at room temperature for 7 days, three hypervariable regions of the bacterial 16S rRNA gene (V1-V2, V3-V4, and V4) were amplicon sequenced. We found that samples collected in two preservatives, Norgen and OMNI, showed the least shift in community composition relative to −80°C standards compared with other storage conditions, and both efficiently inhibited the growth of aerobic and anaerobic bacteria. RNAlater did not prevent bacterial activity and exhibited relatively larger community shift. Although the effect of preservation solution was small compared to intersubject variation, notable changes in microbiota composition were observed, which could create biases in downstream data analysis. When community profiles inferred from different 16S rRNA gene hypervariable regions were compared, we found differential sensitivity of primer sets in identifying overall microbial community and certain bacterial taxa. For example, reads generated by the V4 primer pair showed a higher alpha diversity of the gut microbial community. The degenerate 27f-YM primer failed to detect the majority of Bifidobacteriales. Our data indicate that choice of preservation solution and 16S rRNA gene primer pair are critical determinants affecting gut microbiota profiling. IMPORTANCE Large-scale human microbiota studies require specimens collected from multiple sites and/or time points to maximize detection of the small effects in microbe-host interactions. However, batch biases caused by experimental protocols, such as sample collection, massively parallel sequencing, and bioinformatics analyses, remain critical and should be minimized. This work evaluated the effects of preservation solutions and bacterial 16S rRNA gene primer pairs in revealing human gut microbiota composition. Since notable changes in detecting bacterial composition and abundance were observed among choice of preservatives and primer pairs, a consistent methodology is essential in minimizing their effects to facilitate comparisons between data sets.

No abstract

From this population-based study of a Chinese general population, it was found that smoking, drinking, oral sex, and more sexual partners were associated with alpha human papillomavirus (HPV) infection of the oral cavity. Teeth brushing before sleep was protective for beta/gamma-HPVs.

The role of oral microbiota in head and neck squamous cell carcinoma (HNSCC) is poorly understood. Here we sought to evaluate the association of the bacterial microbiome with host gene methylation and patient outcomes, and to explore its potential as a biomarker for early detection or intervention. Here we performed 16S rRNA gene amplicon sequencing in sixty-eight HNSCC patients across both tissue and oral rinse samples to identify oral bacteria with differential abundance between HNSCC and controls. A subset of thirty-one pairs of HNSCC tumor tissues and the adjacent normal tissues were characterized for host gene methylation profile using bisulfite capture sequencing. We observed significant enrichments of Fusobacterium and Peptostreptococcus in HNSCC tumor tissues when compared to the adjacent normal tissues, and in HNSCC oral rinses when compared to healthy subjects, while ten other bacterial genera were largely depleted. These HNSCC-related bacteria were discriminative for HNSCC and controls with area under the receiver operating curves (AUCs) of 0.84 and 0.86 in tissue and oral rinse samples, respectively. Moreover, Fusobacterium nucleatum abundance in HNSCC cases was strongly associated with non-smokers, lower tumor stage, lower rate of recurrence, and improved disease-specific survival. An integrative analysis identified that enrichment of F. nucleatum was associated with host gene promoter methylation, including hypermethylation of tumor suppressor genes LXN and SMARCA2, for which gene expressions were downregulated in the HNSCC cohort from The Cancer Genome Atlas. In conclusion, we identified a taxonomically defined microbial consortium associated with HNSCC that may have clinical potential regarding biomarkers for early detection or intervention. Host–microbe interactions between F. nucleatum enrichment and clinical outcomes or host gene methylation imply a potential role of F. nucleatum as a pro-inflammatory driver in initiating HNSCC without traditional risk factors, which warrants further investigation for the underlying mechanisms.

No abstract