Combined bacterial and fungal intestinal microbiota analyses: Impact of storage conditions and DNA extraction protocols

<div><p>Background</p><p>The human intestinal microbiota contains a vast community of microorganisms increasingly studied using high-throughput DNA sequencing. Standardized protocols for storage and DNA extraction from fecal samples have been established mostly for bacterial microbiota analysis. Here, we investigated the impact of storage and DNA extraction on bacterial and fungal community structures detected concomitantly.</p><p>Methods</p><p>Fecal samples from healthy adults were stored at -80°C as such or diluted in RNA<i>later</i>® and subjected to 2 extraction protocols with mechanical lysis: the Powersoil® MoBio kit or the International Human Microbiota Standard (IHMS) Protocol Q. Libraries of the 12 samples targeting the V3-V4 16S and the ITS1 regions were prepared using Metabiote® (Genoscreen) and sequenced on GS-FLX-454. Sequencing data were analysed using SHAMAN (<a href="http://shaman.pasteur.fr/" target="_blank">http://shaman.pasteur.fr/</a>). The bacterial and fungal microbiota were compared in terms of diversity and relative abundance.</p><p>Results</p><p>We obtained 171869 and 199089 quality-controlled reads for 16S and ITS, respectively. All 16S reads were assigned to 41 bacterial genera; only 52% of ITS reads were assigned to 40 fungal genera/section. Rarefaction curves were satisfactory in 3/3 and 2/3 subjects for 16S and ITS, respectively. PCoA showed important inter-individual variability of intestinal microbiota largely overweighing the effect of storage or extraction. Storage in RNA<i>later</i>® impacted (downward trend) the relative abundances of 7/41 bacterial and 6/40 fungal taxa, while extraction impacted randomly 18/41 bacterial taxa and 1/40 fungal taxon.</p><p>Conclusion</p><p>Our results showed that RNA<i>later</i>® moderately impacts bacterial or fungal community structures, while extraction significantly influences the bacterial composition. For combined bacterial and fungal intestinal microbiota analysis, immediate sample freezing should be preferred when feasible, but storage in RNA<i>later</i>® remains an option under unfavourable conditions or for concomitant metatranscriptomic analysis; and extraction should rely on protocols validated for bacterial analysis, such as IHMS Protocol Q, and including a powerful mechanical lysis, essential for fungal extraction.</p></div

Workflow of the study.

<p>Comparison of the fungal and bacterial taxonomic diversity of fecal microbiota of 3 healthy individuals (i1, i2, i3) using 2 storage conditions (within two-hours freezing or RNA<i>later</i>® dilution before freezing) and 2 extraction protocols (IHMS Protocol Q and PowerSoil® MoBio kit).</p

Relative abundance of taxa identified from faeces through 16S-sequencing according to storage or extraction conditions.

<p>Bacterial taxa were identified at genus level from fecal samples of 3 healthy individuals (i1, i2 and i3) using two storage conditions (without additive within two-hours freezing or RNA<i>later</i>® dilution before freezing) and two extraction protocols (IHMS Protocol Q and PowerSoil® MoBio kit). 16S rRNA gene ultra-deep-sequencing was performed using 454 technology.</p

Adherence of <i>C. albicans</i> strains overexpressing the Rbt1SL and Rbt1FL proteins (VIF 207 and 208) to human cells.

<p>Yeasts cells were incubated with confluent HeLa cells for 45 minutes (A) or with Caco₂ cells for 30 minutes (B). The percentage of adhesion represents the number of adherent yeasts reported to the number of yeasts in the inoculums. Values given represent mean ± standard deviation (SD) of results of one experiment performed in duplicate and representative of three independent experiments. Pairwise comparisons were made by two-tailed Student’s T-test: significant comparisons are indicated with two asterisks for p value<0.01.</p

<i>In</i><i>vivo</i> localization of Rbt1.

<p>A/ The V5-tagged Rbt1 expressed under the control of the <i>RBT1</i> promoter (VIF210) was detected by immunofluorescence after three different times of hypha induction. Fixed cells were directly treated first with anti-V5 antibodies and then with anti-mouse IgG-Cy3 coupled antibody, and immunofluorescence was observed using an Olympus BX51 microscope. B/ Western blot analysis of proteins solubilized either from the plasma membrane fraction (lanes 1 to 4) or from the cell walls (lanes 5 to 8) of yeast cells (odd numbers) or hyphae (even numbers): lanes 1, 2, 5 and 6, the strain VIF211 expressing the <i>RBT1SL-</i>V5 allele under the control of the <i>ACT1</i> promoter (<i>ACTIp</i>); lanes 3 and 4 the strain VIF106 expressing <i>DCW1</i>-V5 under <i>ACT1p</i> and lanes 7 and 8, the strain VIF105 expressing <i>IFF8</i>-V5 under <i>ACT1p</i>. In lanes 1 to 4, proteins from a membrane-enriched pellet (C_10000g) were solubilized in the presence of 2% SDS; in lanes 5 to 8, the cell wall fraction (C_1000g) was incubated in NaAc buffer + 2U of β-1,6-glucanase for 3 hours at 37°C to solubilize GPI-anchored cell wall proteins. Samples were separated by SDS-PAGE, transferred onto nitrocellulose membrane and immunoblotted with monoclonal anti-V5 antibodies.</p

Aggregation stimulation and aggregation inhibition in the presence of wild type (VTTGVVVVT) and mutated (VTTGNVVVT) peptides added respectively at 2μg.mL^-1 and 20μg.mL^-1.

<p>After filamentation induction by pH and temperature switch (30°C pH5 to 37°C pH7) during 2 hours the strains were further incubated for 24 hours in the presence of the wild type high β-aggregation potential peptide or a mutated peptide. Cells were then examined by light microscopy (×40 magnification).</p

Biofilm formation on Thermanox™ in micro-fermenter of <i>C. albicans</i> strains overexpressing the Rbt1SL and Rbt1FL proteins (VIF 207 and 208).

<p>After an initial immersion period of 30 minutes in the inoculums, plastic slides were further incubated for 40 hours at 37°C in micro-fermenter. A/ Dried weight of each biofilm was measured. The percentages of biomass obtained for the two overexpressing strains were calculated in comparison to those of the wild type control strain which was fixed to 100%. Values given represent mean ± standard deviation (SD) of results of one experiment performed in duplicate and representative of three independent experiments. B/ Pictures of the three biofilms formed on the Thermanox™ lamella after 40 hours. Pairwise comparisons were made by two-tailed Student’s T-test: significant comparison (p value<0.05) are indicated with an asterisk.</p

Rbt1 protein description and sequence similarities.

<p>A/ Schematic representation of the two Rbt1 proteins domain organization with for Rbt1FL from left to right: the signal peptide (black box, aa 1-21); the domain I matching the Flo11 superfamily (dashed box, aa 71-216); the domain II, a Ser/Thr-rich region containing the imperfect repeats 1 and 2 (light grey box, aa 279-396); the domain III (dark grey box, aa 416-555) containing the two 42 amino acid-long repeats (repeat 3) comprising the sequence with a high β-aggregation potential (underlined) and another repeat 2 (containing two additional repeats PESSA); the domain IV (dotted box, aa 556-729) which precedes the GPI anchor addition signal (black box, aa 729-750). The two deletions in the Rbt1SL protein are represented by: Deletion 1 (aa 378-487) and Deletion 2 (aa 612-640). B/ Sequence similarities within the Hwp1 family in the 42 amino acid-long repeat 3. C/ Sequence similarities within the Hwp1 family in the last 60 aa.</p

Major MRI Response in patient 2.

<p>Major MRI response in Patient 2 with disappearance of periachillean tissular infiltration and of talus bone edema. Slight talus contrast enhancement persistance on T1 Gado Fat Sat sequence.</p

Treatment and outcome characteristics of eleven patients with eumycetoma.

<p>B: Bone involvement; CPK: creatinine phosphokinase; CR: Complete Response; D: drainage; I: inflammation; ITZ: itraconazole; J: Joint involvement; KTZ: ketoconazole; M: Muscle involvement; Node involvement; P: pain; PCZ: posaconazole; PF: Primary failure; PR: Partial Response; R: relapse; S: soft tissue involvement; SUV: Standard Uptake Value; V: Visceral involvement; VCZ: voriconazole.</p><p>* Patient 8 only had one post last generation triazole treatment dosage of BG so that evolution couldn’t be assessed.</p><p>Treatment and outcome characteristics of eleven patients with eumycetoma.</p