Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Comparison of Oral Collection Methods for Studies of Microbiota.

Author(s): Vogtmann E, Chen J, Kibriya MG, Amir A, Shi J, Chen Y, Islam T, Eunes M, Ahmed A, Naher J, Rahman A, Barmon B, Knight R, Chia N, Ahsan H, Abnet CC, Sinha R

Publication: Cancer Epidemiol Biomarkers Prev, 2019, Vol. 28, Page 137-143

PubMed ID: 30262598 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the effects of collecting saliva specimens using an expectorant collection device rather than with mouthwash on microbial taxonomic profiles. The stability of microbes in mouthwash specimens after 4 days of storage at ambient temperature was also investigated. Participants included 53 healthy Mayo Clinic employees (≥18 years old, no use of antibiotics or probiotics in 2 weeks prior to collection, no history of pelvic radiation, not currently undergoing chemotherapy) and 50 individuals in the Health Effects of Arsenic Longitudinal Study (HEALS) in Bangladesh from October 2000 to May 2002. Participants were asked to refrain from eating or smoking at least 20 minutes prior to saliva collection by expectoration using the OMNIgene ORAL OM-505 Collection Device and were then asked to swish 10 mL of mouthwash for 5 seconds, gargle for 5 seconds, repeat the swish and gargle for a total of 30 seconds, and then spit the mouthwash into a collection cup. The OMNIgene tube was shaken, incubated at 50°C for 1 hour in a water bath, and then frozen at -80°C. Two aliquots were created from the mouthwash sample: one was frozen immediately at -80°C and the other remained at room temperature for 96 hours (4 days) and then frozen at -80°C. Specimens were shipped on dry ice to the University of California (San Diego, CA), thawed at 4°C, and kept on ice during plating. A wooden swab was dipped into each aliquot for DNA extraction using the MO-BIO PowerMag Soil DNA Isolation Kit. The V4 region of the 16S rRNA gene was amplified by PCR and sequencing was performed using the Illumina HiSeq.

   Summary of Findings:

   The taxonomic profiles were significantly different between the Mayo Clinic and HEALS specimens and between the two collection methods (P< 0.001); however, inter-subject variability represented a higher proportion of microbial variability for all measures of diversity in both Mayo Clinic and HEALS specimens compared to collection method (mouthwash or expectoration) or day of freezing (immediately or after 4 days at ambient temperature). The relative abundance of Spirochaetes was greater in the HEALS cohort for both expectoration and mouthwash specimens compared to Mayo Clinic specimens while the relative abundance of Actinobacteria in expectoration specimens was greater in the Mayo Clinic cohort than the HEALS cohort but were similar in the mouthwash specimens. Comparability of relative abundance of the top 25 genera in mouthwash specimens compared to expectoration specimens in both the Mayo Clinic and HEALS cohorts was generally low (R<0.75), but a few were considered acceptable (Bacteroidetes and observed suboperational taxonomic units (sOTU), R=0.77 for both and Proteobacteria, R=0.81). In contrast, higher levels of the phylum Firmicutes were observed in mouthwash specimens than expectorated specimens. The stability in relative abundance of the top 25 genera was high for mouthwash specimens after 4 days at ambient temperature (r ≥0.75) in both the Mayo Clinic and HEALS cohorts.

   Biospecimens

   • Bodily Fluid - Saliva

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Time at room temperature	4 days 0 days
   Biospecimen Acquisition	Method of fluid acquisition	Swishing Expectoration

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