biostudies-arrayexpressKavana Bywater-BrennaClostridioides difficilehttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15646This experiment was performed largely to capture the dynamic transcriptional changes occurring in C. difficile when cultured within a 9 species synthetic gut communal consortium over a 48 h time series when cultured as a biofilm in vitro.biostudies-arrayexpressSample Collection - For the multispecies microbiota community, overnight cultures of each species were grown in Schaedler anaerobe broth (SAB)supplemented with 2 g/L L-cysteine and 0.005 g/L vitamin K. Overnight cultures were mixed so that each species had a final concentration of 0.1 OD600 within the mixture. For C. difficile monoculture controls, overnight cultures were also diluted to a final concentration of 0.1 OD600. 2 mL aliquots of these cultures were then pipetted into pre-reduced 12-well tissue culture treated polystyrene plates (Falcon®, Corning, USA). Biofilms were washed at the desired timepoints with 1 mL PBS, and disrupted with 750 µL LETS buffer (0.1M LiCl, 0.01M Na2EDTA, 0.01M Tris-HCl (pH 7.5), 0.2% SDS) for subsequent TRIzol-based extraction.Sequencing - The sequencing libraries were multiplexed and loaded on the flow cell on the Illumina NovaSeq 6000 instrument according to manufacturer’s instructions. The samples were sequenced using a 2x150 Pair-End (PE) configuration v1.5. Image analysis and base calling were conducted by the NovaSeq Control Software v1.7 on the NovaSeq instrument. Raw sequence data (.bcl files) generated from Illumina NovaSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq program version 2.20. One mismatch was allowed for index sequence identification. This was performed by Genewiz (Azenta)Library Construction - RNA sequencing library preparation was prepared using NEBNext rRNA Depletion Kit (Bacteria) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina following manufacturer’s instructions (NEB, Ipswich, MA, USA). Briefly, rRNA was depleted with NEBNext rRNA Depletion Kit (Bacteria). rRNA depleted RNAs were fragmented. First strand and second strand cDNA were subsequently synthesized. The second strand of cDNA was marked by incorporating dUTP during the synthesis. cDNA fragments were adenylated at 3’ends, and indexed adapter was ligated to cDNA fragments. Limited cycle PCR was used for library amplification. The dUTP incorporated into the cDNA of the second strand enabled its specific degradation to maintain strand specificity. This was performed by Genewiz (Azenta)Nucleic Acid Extraction - Samples were homogenised in 2 mL Lysing Matrix B (MP Biomedicals, USA) tubes, by bead beating for 6 cycles at 6.5 m/s for 40 seconds in a FastPrep-24-5G (MP Biomedicals, USA). Samples were kept on ice for 3 min between each cycle. The samples were subsequently spun at 15,000 x g for 10 min, and the supernatants were transferred into 2 mL nuclease-free Biosphere screw-cap tubes (Sarstedt Ltd., Germany) prior to extraction. A 1 mL aliquot of TRIzol Reagent (Invitrogen, USA) was added to each supernatant, followed by a 5 min incubation at RT. 200 µL chloroform (Sigma-Aldrich, USA) was added to each sample and briefly vortexed and incubated again at RT for 5 min. Following this incubation, the samples were centrifuged at 12,000 x g at 4°C for 15 min. The resulting upper aqueous phases were then removed to new tubes, 500 µL isopropanol was added to each tube, and the samples were incubated at RT for 10 min followed by a 10 min centrifugation at 12,000 x g at 4 °C. The supernatants were discarded, and the pellets were allowed to air-dry prior to resuspending them in 30 µL RNase-free water. RNA concentrations were then quantified using Qubit RNA BR assay kits (Thermo Fisher Scientific, USA). RNA samples were DNase treated using the Turbo DNA-free kit (Thermo Fisher Scientific, USA), following the rigorous treatment according to the manufacture’s protocol. Briefly, samples were, where necessary, diluted to a final concertation of 200 ng/µL in a total reaction volume of 50 µL. Each 50 µL reaction contained 5 µL 10x Turbo DNase Buffer and 1 µL DNase. Upon addition of DNase, samples were gently mixed, and incubated at 37 °C for 30 min. 10 µL DNase Inactivation Reagent was added to each tube, gently mixed by flicking and incubated at RT for a further 5 min. The tubes were flicked at 2 min intervals to ensure the inactivation reagents remained well dispersed in solution. Finally, the samples were centrifuged for 1.5 min at 10,000 x g. The supernatants were then moved to new nuclease-free tubes. To further remove contaminants such as residual DNA, proteins or carbohydrates, samples were treated with lithium chloride. Here, LiCl Precipitation Solution (Invitrogen, USA) was added to each DNase-treated RNA sample at a final concentration of 2.5 M. The samples were then incubated at –20 °C for 30 min, prior to centrifugation at 14,000 RPM at 4 °C for 15 min. The supernatants were discarded, the pellets washed with 200 µL ice-cold 70 % ethanol and were then subsequently centrifuged for 5 min at 14,000 RPM, before air-dying the pellets and resuspending them in 20 µL nuclease-free water.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Reads were trimmed to remove adapter contaminants using Trimmomatic (Version 0.39)92, based on a list of common adapter sequences. Unpaired reads were ignored. FastQC was rerun to ensure satisfactory quality of output files. Following trimming, reads were aligned to the appropriate genomes (concatenated in the case of synthetic community samples) using HISAT-2 (Version 2.1.0). As reads came from strictly bacterial cultures, the in-built splice-awareness setting was turned off in HISAT-2. Unpaired reads were ignored during this step. Samtools were used to convert the resulting SAM files to BAM files, and were subsequently sorted and indexes. Unmapped reads were discarded during this step. Gene counts from the resulting BAM files were generated using Libinorm which was run in HTseq-compatible mode. For this, appropriate genome annotation files were used, concatenated where necessary.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000Clostridioides difficile is a major cause of hospital-associated diarrhoea worldwide. The intricate interactions between C. difficile and the resident gut microbiota play a crucial role in determining the outcome of C. difficile infection (CDI), although the molecular mechanisms underlying many C. difficile -commensal interactions are not understood. Here we show that selected Bacteroides species can inhibit C. difficile growth within mixed biofilms. A transcriptomic analysis of C. difficile-Bacteroides biofilms showed significant metabolic shifts, with distinct changes in carbohydrate and amino acid metabolism and, interestingly, a downregulation of C. difficile toxin gene expression. A significant reduction in C. difficile toxin production was evident in C. difficile - Bacteroides cocultures, irrespective of the extent of C. difficile growth inhibition. Notably, Stickland fermentation of proline, which is known to repress toxin synthesis, was upregulated in C. difficile , while proline synthesis was induced in the cocultured species B. vulgatus and B. dorei . Furthermore, upregulation of proline reductase pathways and consequent toxin repression were evident within a synthetic 9-species gut commensal biofilm community containing multiple Bacteroides spp. Thus, leveraging multiomics approaches, we demonstrate a potential cross-feeding mechanism where proline produced by B. dorei and B. vulgatus is utilised by C. difficile through Stickland fermentation to drive toxin repression. Our study reveals a new mechanism of microbiota-mediated control of a key virulence factor involved in C. difficile pathogenesis while enabling pathogen co-existence within a polymicrobial commensal community. <h4>Importance</h4> C. difficile infection, characterised by severe diarrhoea and colitis, has a significant impact on healthcare settings globally due to the high rates of recurrence. CDI is closely associated with the gut microbiota status and the use of antibiotics, yet the mechanistic basis of interactions between the causative bacterium C. difficile and individual gut commensal species remains poorly defined. Here, we demonstrate inhibitory effects of Bacteroides species on C. difficile through nutrient competition and a cross-feeding mechanism between these abundant gut commensals and this pathogen which blocks expression of key C. difficile virulence factors. Our findings offer insights into the effective design of microbiota consortia to prevent and treat CDI.RNA-seq of total RNAClostridioides difficileBacteroides-driven metabolic remodelling suppresses Clostridioides difficile toxin expression in mixed biofilm communitiesKavana Bywater-BrennaKavana Kalea Bywater-Brenna, Simran Kaur Aulakh, Kiran Raosaheb Patil, Niranjan Nagarajan and Meera UnnikrishnanMeera UnnikrishnanfalseTemporal RNASeq of C. difficile cultured within synthetic multispecies biofilm relative to C. difficile monoculture biofilms.This experiment was performed largely to capture the dynamic transcriptional changes occurring in C. difficile when cultured within a 9 species synthetic gut communal consortium over a 48 h time series when cultured as a biofilm in vitro.2026-09-23T00:00:00Z2026-09-23T01:00:45.88Z2025-09-30T23:10:54.491ZE-MTAB-15646ERP180844EFO_0002944EFO_0004170EFO_0009653EFO_0005518EFO_0003816EFO_000418410.1101/2025.09.03.674004