Comparison of Oral Collection Methods for Studies of Microbiota

Abstract

Background: A number of cohort studies have collected Scope mouthwash samples by mail, which are being used for microbiota measurements. We evaluated the stability of Scope mouthwash samples at ambient temperature and determined the comparability of Scope mouthwash with saliva collection using the OMNIgene ORAL Kit.

Methods: Fifty-three healthy volunteers from Mayo Clinic and 50 cohort members from Bangladesh provided oral samples. One aliquot of the OMNIgene ORAL and Scope mouthwash were frozen immediately and one aliquot of the Scope mouthwash remained at ambient temperature for 4 days and was then frozen. DNA was extracted and the V4 region of the 16S rRNA gene was PCR amplified and sequenced using the HiSeq. Intraclass correlation coefficients (ICC) were calculated.

Results: The overall stability of the Scope mouthwash samples was relatively high for alpha and beta diversity. For example, the meta-analyzed ICC for the Shannon index was 0.86 (95% confidence interval, 0.76-0.96). Similarly, the ICCs for the relative abundance of the top 25 genera were generally high. The comparability of the two sample types was relatively low when measured using ICCs, but were increased by using a Spearman correlation coefficient (SCC) to compare the rank order of individuals.

Conclusions: Overall, the Scope mouthwash samples appear to be stable at ambient temperature, which suggests that oral rinse samples received by the mail can be used for microbial analyses. However, Scope mouthwash samples were distinct compared with OMNIgene ORAL samples.

Impact: Studies should try to compare oral microbial metrics within one sample collection type.

FIGURE 1.

Stacked barplot of the relative abundances at the phylum, family, and genus level for OMNIgene ORAL (OMNI) and Scope mouthwash samples (both Day 0 and Day 4) from Mayo Clinic (M) and Bangladesh (B). Using the PERMANOVA test for the Bray-Curtis difference, the taxonomic profiles for the two populations were statistically different for both the OMNIgene ORAL and Scope mouthwash collections (P < 0.001).

FIGURE 2.

Percent of microbial variability explained by subject (black), sample collection method (grey), and day of freezing (white) was calculated using an adjusted distance-based coefficient of determination R² for beta-diversity estimates from unweighted UniFrac, generalized UniFrac, weighted UniFrac, and Bray-Curtis (BC) distance for Mayo Clinic and Bangladesh samples.

FIGURE 3.

Stability of Scope mouthwash samples incubated at ambient temperature for four days (Day 4) compared to samples frozen immediately (Day 0) for the relative abundance of four phyla, two alpha-diversity metrics, and five beta-diversity matrices using intraclass correlation coefficients for Mayo Clinic and Bangladesh samples.

FIGURE 4.

Comparability of the immediately frozen Scope mouthwash to OMNIgene ORAL kit samples for the relative abundance of four phyla, two alpha-diversity metrics, and five beta-diversity matrices using intraclass correlation coefficients (3A) and Spearman correlations (3B) for Mayo Clinic and Bangladesh samples.