Accuri cflow software

Samples were centrifuged at 10,000×g for 3 min and supernatant discarded. Pelleted sample was fixed for 4 h at 4 °C with 4% (w/v) filtered paraformaldehyde (pH 7.2) in a ratio of 1:3 (v/v). Samples were washed twice with filtered PBS and resuspended in 600 μL of a mixture of PBS/ethanol (1:1, v/v) and then stored at − 20 °C for up to 3 months. Hybridisation was carried out as described in Rycroft et al. (2001a (link), b (link)) using genus and group specific 16S rRNA-targeted oligonucleotide probes (MWG Biotech, Ebersberg, Germany).
The sample probes used were Bif164 (Langendijk et al. 1995 (link)), Bac303 (Manz et al. 1996 (link)), Lab158 (Harmsen et al. 2000 (link)), Ato291³, Prop853 (Walker et al. 2005 (link)), Erec482 (Franks et al. 1998 (link)), Rrec584 (Walker et al. 2005 (link)), Fprau655 (Hold et al. 2003 (link)), Chis150 (Franks et al. 1998 (link)), shown in Additional file 1: Table S1. Samples were screened using a flow cytometer (Accuri C6, BD Biosciences, USA) with Accuri CFlow software.

Microbial load of the study cohort was measured as described previously^{7 (link)}. Briefly, 200–250 mg of frozen (−80 °C) fecal aliquots was diluted in saline solution (0.85% NaCl; VWR International) and filtered using a sterile syringe filter (a pore size of 5 µm; Sartorius Stedim Biotech). Next, 1 ml of the microbial cell suspension obtained was stained with 1 µl of SYBR Green I (1:100 dilution in DMSO; Thermo Fisher Scientific) and incubated for 15 min in the dark at 37 °C. The flow cytometry analysis was performed using a C6 Accuri flow cytometer (BD Biosciences) according to Prest et al.^{11 (link)}. Fluorescence events were monitored using the FL1 533/30-nm and FL3 > 670-nm optical detectors. The BD Accuri CFlow software was used to gate and separate the microbial fluorescence events on the FL1/FL3 density plot from the fecal sample background. A threshold value of 2,000 was applied on the FL1 channel. Based on the exact weight of the aliquots analyzed, cell counts were converted to microbial loads per gram of fecal material.

The bacterial cell count in the inoculum was measured by flow cytometry (BD Accuri C6 flow cytometer, BD Biosciences, Franklin Lakes, NJ) as previously described^{36 (link)}. Samples (200 μL) were transferred to a sterile Eppendorf tube and incubated at 35 °C for 10 min prior to staining with SYBR Green I (2 μL of 100× stock solution in 200 μL sample), vortexed, and then incubated again at 35 °C for 10 min. Samples (200 μL) were then transferred to a 96-well plate for cell counting. Concurrently, another 200 µL of the samples were stained with propidium iodide (2 μL of 100× stock solution) in combination with SYBR Green I (2 μL of 100× stock solution) to find live and dead (membrane-compromised) cells according to the same protocol used for total bacterial cell count. A flow cytometer equipped with a 50 mW laser having a fixed emission wavelength of 488 nm was used to measure the cell count. Fluorescence intensity was collected at FL1 = 533 ± 30 nm, FL3 > 670 nm, sideward and forward scattered light intensities were obtained as well. Electronic gating was used to select SYBR green I as well as propidium iodide and SYBR Green I staining labeled signals for quantifying total bacterial as well as live and dead cells. All data were processed with the BD Accuri CFlow® software. Unless specified otherwise, the bacterial counts represent the live cell number.