Comparison of fecal and oral collection methods for studies of the human microbiota in two Iranian cohorts

Abstract

Background To initiate fecal and oral collections in prospective cohort studies for microbial analyses, it is essential to understand how field conditions and geographic differences may impact microbial communities. This study aimed to investigate the impact of fecal and oral sample collection methods and room temperature storage on collection samples for studies of the human microbiota. Results We collected fecal and oral samples from participants in two Iranian cohorts located in rural Yazd (n = 46) and urban Gonbad (n = 38) and investigated room temperature stability over 4 days of fecal (RNAlater and fecal occult blood test [FOBT] cards) and comparability of fecal and oral (OMNIgene ORAL kits and Scope mouthwash) collection methods. We calculated interclass correlation coefficients (ICCs) based on 3 alpha and 4 beta diversity metrics and the relative abundance of 3 phyla. After 4 days at room temperature, fecal stability ICCs and ICCs for Scope mouthwash were generally high for all microbial metrics. Similarly, the fecal comparability ICCs for RNAlater and FOBT cards were high, ranging from 0.63 (95% CI: 0.46, 0.75) for the relative abundance of Firmicutes to 0.93 (95% CI: 0.89, 0.96) for unweighted Unifrac. Comparability ICCs for OMNIgene ORAL and Scope mouthwash were lower than fecal ICCs, ranging from 0.55 (95% CI: 0.36, 0.70) for the Shannon index to 0.79 (95% CI: 0.69, 0.86) for Bray-Curtis. Overall, RNAlater, FOBT cards and Scope mouthwash were stable up to 4 days at room temperature. Samples collected using FOBT cards were generally comparable to RNAlater while the OMNIgene ORAL were less similar to Scope mouthwash. Conclusions As microbiome measures for feces samples collected using RNAlater, FOBT cards and oral samples collected using Scope mouthwash were stable over four days at room temperature, these would be most appropriate for microbial analyses in these populations. However, one collection method should be consistently since each method may induce some differences.

Stability of Oral and Fecal Microbiome at Room Temperature: Impact on Diversity

“…Previous studies used Scope® oral wash to preserve oral microbiome samples (Vogtmann et al ., 2019; Yano et al ., 2020), and its stability at room temperature was already verified (Vogtmann et al ., 2019; Wu et al ., 2021); however, it is not easily found in Europe. As chlorhexidine has been commonly used in many clinical trials where effective results have been proven in reducing the proliferation of bacterial species (Eick et al ., 2011; James et al ., 2017; Ben-Knaz Wakshlak, Pedahzur and Avnir, 2019; Brookes et al ., 2020; Sedghi et al ., 2021; Xiang, Rojo and Prados-Frutos, 2021), we opted for Lacer® Chlorhexidine oral wash to preserve the samples.…”

“…The room temperature stability of other fecal microbiome collection methods has been proven for FTA cards at 8 weeks (Song et al ., no date), OMNIgene Gut Kit for 3 days (Choo, Leong and Rogers, 2015) and 8 weeks (Park et al ., 2020; Song et al ., no date), FOBT for 3 (Dominianni et al ., 2014) and 4 days (Sinha et al ., 2016; Wu et al ., 2021) and 1 week (Gudra et al ., 2017; Byrd et al ., 2019), RNAlater for 3 (Choo, Leong and Rogers, 2015; Roberto Flores et al ., 2015), 4 (Sinha et al ., 2016; Wu et al ., 2021) and 7 days (Roberto Flores et al ., 2015; Byrd et al ., 2019) and 8 weeks (Park et al ., 2020).…”

“…Regarding the oral microbiome, previous studies used Scope® oral wash (mainly composed of Alcohol, Domiphen Bromide and Cetylpyridinium Chloride) to preserve oral microbiome samples (Vogtmann et al ., 2019; Yano et al ., 2020), as it has been demonstrated that samples preserved with Scope are stable in terms of alpha and beta diversity up to 4 days at RT (Vogtmann et al ., 2019; Wu et al ., 2021). However, as this solution is not easily found in Europe, Chlorhexidine oral wash (Lacer®) was used.…”