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. 2022 Aug 12;14(16):3315.
doi: 10.3390/nu14163315.

Epidemiological Studies of Children's Gut Microbiota: Validation of Sample Collection and Storage Methods and Microbiota Analysis of Toddlers' Feces Collected from Diapers

Hazuki Tamada  1 , Yuki Ito  1 , Takeshi Ebara  1 , Sayaka Kato  1 , Kayo Kaneko  1 , Taro Matsuki  1 , Mayumi Sugiura-Ogasawara  2 , Shinji Saitoh  3 , Michihiro Kamijima  1
Affiliations

Epidemiological Studies of Children's Gut Microbiota: Validation of Sample Collection and Storage Methods and Microbiota Analysis of Toddlers' Feces Collected from Diapers

Hazuki Tamada et al. Nutrients. .

Abstract

The composition of human gut microbiota influences human health and disease over the long term. Since the flora in specimens can easily change at ambient temperature outside the body, epidemiological studies need feasible methods of stool specimen collection and storage to be established. We aimed to validate two methods: feces frozen-stored in tubes containing guanidine thiocyanate solution for two months after collection (Method B), and feces excreted in diapers and frozen-stored (Method C). Validation was by comparison with a gold standard Method A. Bacterial flora of five adults were sampled and stored by all three methods. Bacterial composition was examined by amplicon sequencing analysis. Bland-Altman analyses showed that Methods B and C might change relative abundances of certain bacterial flora. Thereafter, we analyzed the bacterial flora of 76 toddlers (two age groups) in stools sampled and processed by Method C. The diversity indices of toddlers' flora were less than those of adults. The relative abundance of some bacteria differed significantly between children aged 1.5 and 3 years. The specimen collection and storage methods validated in this study are worth adopting in large-scale epidemiological studies, especially for small children, provided the limited accuracy for some specific bacteria is understood.

Keywords: diaper; epidemiology; feces; gut microbiome; toddlers.

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Conflict of interest statement

The authors declare no conflict of interest. The sponsors had no role in the design, execution, interpretation, or writing of the study.

Figures

Figure 1
Figure 1
Experimental overview of Research I. For Methods A and B, stool samples were collected using commercial collection tubes. For Method C, urine and stool samples were applied to disposable diapers. For Methods B and C, the samples were stored at −80 °C for 2 months.
Figure 2
Figure 2
Research I: Fecal bacterial composition in five adult volunteers. Bacterial composition at the (a) phylum and (b) genus levels. Bacteria detected at a relative abundance of less than 1% were grouped together as Others. † See Table S1 or Table S5 for the bacterial taxonomy name corresponding to each ID.
Figure 3
Figure 3
Research I: Diversity indices and the Firmicutes/Bacteroidetes ratio of the fecal bacterial community in five adult volunteers. (a) Diversity indices and the (b) Firmicutes/Bacteroidetes ratio in each method.
Figure 4
Figure 4
Research I: Bland–Altman plots of the relative abundance of fecal bacteria at the phylum level obtained by each method from five adult volunteers. Comparisons between Methods A–B (left) and Methods A–C (right). The relative abundance of bacteria in each specimen was plotted. (a) Their maximum abundance in any of the specimens was ≥10%; (b) ≥1% and <10%; (c) <1%. Solid lines indicate the means of the differences between the two test values, dotted lines indicate the limits of agreement (the mean of the difference ± 1.96 × standard deviation), and the gray shaded area indicates the 95% confidence interval of the limits of agreement. For Methods A and B, stool samples were collected using commercial collection tubes. For Method C, urine and stool samples were applied to disposable diapers. For Methods B and C, the samples were stored at −80 °C for 2 months.
Figure 5
Figure 5
Research I: Bland–Altman plots of the relative abundance of fecal bacteria at the genus level obtained by each method from five adult volunteers. Comparisons between Methods A–B (left) and Methods A–C (right). The relative abundance of bacteria in each specimen was plotted. (a) Their maximum abundance in any of the specimens was ≥10%; (b) ≥1% and <10%; (c) ≥0.1% and <1%; (d) <0.1%. Solid lines indicate the means of the differences between the two test values, dotted lines indicate the limits of agreement (the mean of the difference ± 1.96 × standard deviation), and the gray shaded area indicates the 95% confidence interval of the limits of agreement. For Methods A and B, stool samples were collected using commercial collection tubes. For Method C, urine and stool samples were applied to disposable diapers. For Methods B and C, the samples were stored at −80 °C for 2 months.
Figure 6
Figure 6
Research I: Heatmap of the gut microbiota composition at the genus level in five adult volunteers. Heatmap of log-transformed values of the relative abundance at the genus level in each method of Research I, using Ward’s method for clustering.
Figure 7
Figure 7
Research II: Fecal bacterial composition at the phylum and genus levels in 76 toddlers. (a) Phylum and (b) genus levels. Bacteria detected at less than 1% of their relative abundance were grouped together as Others. These were sorted by age and mode of delivery. † See Table S1 or Table S5 for the bacterial taxonomy name corresponding to each ID.
Figure 8
Figure 8
Box plots of the bacteria listed in Table 5 that showed significant different relative abundances between the two age groups (1.5 years and 3 years). The depicted bacteria are those whose abundance differences between Methods A–C were within the limits of agreement in all the specimens (See Table 5). Bold lines in the middle of the box are the medians, the top of the box is the 3rd quartile, the bottom of the box is the 1st quartile, the upper whiskers are the largest data less than or equal to “3rd quartile + 1.5 × (3rd quartile − 1st quartile)” and the lower whiskers are the smallest data equal to “1st quartile − 1.5 × (3rd quartile − 1st quartile)”. See Tables S1–S6 for the bacterial taxonomy name corresponding to each ID.
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