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16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice.

Walker AW, Martin JC, Scott P, Parkhill J, Flint HJ, Scott KP - Microbiome (2015)

Bottom Line: Here, several widely used molecular techniques were compared to establish the optimal methods to assess the bacterial composition in faecal samples from babies, before weaning.Using the 16S rRNA gene sequencing approach, however, we found that the detection of bifidobacteria in particular crucially depended on the optimisation of the DNA extraction method, and the choice of primers used to amplify the V1-V3 regions of 16S rRNA genes prior to subsequent sequence analysis.This work emphasises the importance of sample processing methodology to downstream sequencing results and illustrates the value of employing multiple approaches for determining microbiota composition.

View Article: PubMed Central - PubMed

Affiliation: Microbiology Group, Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen, AB21 9SB UK ; Pathogen Genomics Group, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire CB10 1SA UK.

ABSTRACT

Background: Characterisation of the bacterial composition of the gut microbiota is increasingly carried out with a view to establish the role of different bacterial species in causation or prevention of disease. It is thus essential that the methods used to determine the microbial composition are robust. Here, several widely used molecular techniques were compared to establish the optimal methods to assess the bacterial composition in faecal samples from babies, before weaning.

Results: The bacterial community profile detected in the faeces of infants is highly dependent on the methodology used. Bifidobacteria were the most abundant bacteria detected at 6 weeks in faeces from two initially breast-fed babies using fluorescent in situ hybridisation (FISH), in agreement with data from previous culture-based studies. Using the 16S rRNA gene sequencing approach, however, we found that the detection of bifidobacteria in particular crucially depended on the optimisation of the DNA extraction method, and the choice of primers used to amplify the V1-V3 regions of 16S rRNA genes prior to subsequent sequence analysis. Bifidobacteria were only well represented among amplified 16S rRNA gene sequences when mechanical disruption (bead-beating) procedures for DNA extraction were employed together with optimised "universal" PCR primers. These primers incorporate degenerate bases at positions where mismatches to bifidobacteria and other bacterial taxa occur. The use of a DNA extraction kit with no bead-beating step resulted in a complete absence of bifidobacteria in the sequence data, even when using the optimised primers.

Conclusions: This work emphasises the importance of sample processing methodology to downstream sequencing results and illustrates the value of employing multiple approaches for determining microbiota composition.

No MeSH data available.


Related in: MedlinePlus

Longitudinal bacterial profile of two babies (pre-weaning), comparing FISH and 16S rRNA gene sequencing data. a, b—sequencing data (27f-Mix primer set); c, d—FISH data. a, c Baby N-BF, natural birth, breast-fed only; b, d Baby C-MF, C-section, one bottle formula/day introduced from 5 weeks. FISH probes used were Eub338 (total bacterial count), Erec482 (Lachnospiraceae), Fprau645 (F. prausnitzii group of the Ruminococcaceae), Bif164 (Bifidobacterium genus), Rum730 (Rfla729 + Rbro730) (Ruminococcus flavefaciens and R. bromii subclusters of the Ruminococcaceae), Prop853 (Veillonellaceae), Bac303 (Bacteroides-Prevotella group), LAB158 (Lactobacillaceae and Enterococcaceae) and EntD (Enterobacteriaceae). The same colouring scheme has been used to illustrate overlap between bacterial taxa identified using the two methods
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Fig3: Longitudinal bacterial profile of two babies (pre-weaning), comparing FISH and 16S rRNA gene sequencing data. a, b—sequencing data (27f-Mix primer set); c, d—FISH data. a, c Baby N-BF, natural birth, breast-fed only; b, d Baby C-MF, C-section, one bottle formula/day introduced from 5 weeks. FISH probes used were Eub338 (total bacterial count), Erec482 (Lachnospiraceae), Fprau645 (F. prausnitzii group of the Ruminococcaceae), Bif164 (Bifidobacterium genus), Rum730 (Rfla729 + Rbro730) (Ruminococcus flavefaciens and R. bromii subclusters of the Ruminococcaceae), Prop853 (Veillonellaceae), Bac303 (Bacteroides-Prevotella group), LAB158 (Lactobacillaceae and Enterococcaceae) and EntD (Enterobacteriaceae). The same colouring scheme has been used to illustrate overlap between bacterial taxa identified using the two methods

Mentions: The two babies had very different bacterial profiles, and it took between 3 and 7 weeks for the infant microbiota to stabilise. Although the panel of FISH probes had previously been shown to cover 80 % of the microbial species present in adult faecal samples [36], more than 50 % of the bacteria were unidentified in early samples from baby N-BF (Fig. 3c; Additional file 1: Figure S5). The population of bifidobacteria increased steadily to the 14-week time point, when approximately 60 % of the bacteria present in baby N-BF were bifidobacteria and Bacteroides populations remained undetectable (Fig. 3a, c). In contrast, with baby C-MF, the maximum population of bifidobacteria (>60 %) was detected at the 2- and 4-week time points (Fig. 3b, d; Additional file 1: Figure S5). During the transitional 5-week period between the introduction of formula-feeding and the complete withdrawal of breast milk, the bifidobacteria population decreased finally representing less than 10 % of the total microbiota (Additional file 1: Figure S5), while Bacteroides species became prevalent by 9 weeks and were maintained at >50 % of the total population until just before weaning. These findings are broadly consistent with previous studies of formula-fed versus breast-fed infants [7, 9, 37, 38].Fig. 3


16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice.

Walker AW, Martin JC, Scott P, Parkhill J, Flint HJ, Scott KP - Microbiome (2015)

Longitudinal bacterial profile of two babies (pre-weaning), comparing FISH and 16S rRNA gene sequencing data. a, b—sequencing data (27f-Mix primer set); c, d—FISH data. a, c Baby N-BF, natural birth, breast-fed only; b, d Baby C-MF, C-section, one bottle formula/day introduced from 5 weeks. FISH probes used were Eub338 (total bacterial count), Erec482 (Lachnospiraceae), Fprau645 (F. prausnitzii group of the Ruminococcaceae), Bif164 (Bifidobacterium genus), Rum730 (Rfla729 + Rbro730) (Ruminococcus flavefaciens and R. bromii subclusters of the Ruminococcaceae), Prop853 (Veillonellaceae), Bac303 (Bacteroides-Prevotella group), LAB158 (Lactobacillaceae and Enterococcaceae) and EntD (Enterobacteriaceae). The same colouring scheme has been used to illustrate overlap between bacterial taxa identified using the two methods
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4482049&req=5

Fig3: Longitudinal bacterial profile of two babies (pre-weaning), comparing FISH and 16S rRNA gene sequencing data. a, b—sequencing data (27f-Mix primer set); c, d—FISH data. a, c Baby N-BF, natural birth, breast-fed only; b, d Baby C-MF, C-section, one bottle formula/day introduced from 5 weeks. FISH probes used were Eub338 (total bacterial count), Erec482 (Lachnospiraceae), Fprau645 (F. prausnitzii group of the Ruminococcaceae), Bif164 (Bifidobacterium genus), Rum730 (Rfla729 + Rbro730) (Ruminococcus flavefaciens and R. bromii subclusters of the Ruminococcaceae), Prop853 (Veillonellaceae), Bac303 (Bacteroides-Prevotella group), LAB158 (Lactobacillaceae and Enterococcaceae) and EntD (Enterobacteriaceae). The same colouring scheme has been used to illustrate overlap between bacterial taxa identified using the two methods
Mentions: The two babies had very different bacterial profiles, and it took between 3 and 7 weeks for the infant microbiota to stabilise. Although the panel of FISH probes had previously been shown to cover 80 % of the microbial species present in adult faecal samples [36], more than 50 % of the bacteria were unidentified in early samples from baby N-BF (Fig. 3c; Additional file 1: Figure S5). The population of bifidobacteria increased steadily to the 14-week time point, when approximately 60 % of the bacteria present in baby N-BF were bifidobacteria and Bacteroides populations remained undetectable (Fig. 3a, c). In contrast, with baby C-MF, the maximum population of bifidobacteria (>60 %) was detected at the 2- and 4-week time points (Fig. 3b, d; Additional file 1: Figure S5). During the transitional 5-week period between the introduction of formula-feeding and the complete withdrawal of breast milk, the bifidobacteria population decreased finally representing less than 10 % of the total microbiota (Additional file 1: Figure S5), while Bacteroides species became prevalent by 9 weeks and were maintained at >50 % of the total population until just before weaning. These findings are broadly consistent with previous studies of formula-fed versus breast-fed infants [7, 9, 37, 38].Fig. 3

Bottom Line: Here, several widely used molecular techniques were compared to establish the optimal methods to assess the bacterial composition in faecal samples from babies, before weaning.Using the 16S rRNA gene sequencing approach, however, we found that the detection of bifidobacteria in particular crucially depended on the optimisation of the DNA extraction method, and the choice of primers used to amplify the V1-V3 regions of 16S rRNA genes prior to subsequent sequence analysis.This work emphasises the importance of sample processing methodology to downstream sequencing results and illustrates the value of employing multiple approaches for determining microbiota composition.

View Article: PubMed Central - PubMed

Affiliation: Microbiology Group, Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen, AB21 9SB UK ; Pathogen Genomics Group, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire CB10 1SA UK.

ABSTRACT

Background: Characterisation of the bacterial composition of the gut microbiota is increasingly carried out with a view to establish the role of different bacterial species in causation or prevention of disease. It is thus essential that the methods used to determine the microbial composition are robust. Here, several widely used molecular techniques were compared to establish the optimal methods to assess the bacterial composition in faecal samples from babies, before weaning.

Results: The bacterial community profile detected in the faeces of infants is highly dependent on the methodology used. Bifidobacteria were the most abundant bacteria detected at 6 weeks in faeces from two initially breast-fed babies using fluorescent in situ hybridisation (FISH), in agreement with data from previous culture-based studies. Using the 16S rRNA gene sequencing approach, however, we found that the detection of bifidobacteria in particular crucially depended on the optimisation of the DNA extraction method, and the choice of primers used to amplify the V1-V3 regions of 16S rRNA genes prior to subsequent sequence analysis. Bifidobacteria were only well represented among amplified 16S rRNA gene sequences when mechanical disruption (bead-beating) procedures for DNA extraction were employed together with optimised "universal" PCR primers. These primers incorporate degenerate bases at positions where mismatches to bifidobacteria and other bacterial taxa occur. The use of a DNA extraction kit with no bead-beating step resulted in a complete absence of bifidobacteria in the sequence data, even when using the optimised primers.

Conclusions: This work emphasises the importance of sample processing methodology to downstream sequencing results and illustrates the value of employing multiple approaches for determining microbiota composition.

No MeSH data available.


Related in: MedlinePlus