biostudies-arrayexpressKavana Bywater-BrennaPhocaeicola vulgatushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15641The aim of this study was to investigate transcriptomic changes in Clostridioides difficile when cultured as monoculture biofilms or as co-culture biofilms with either Bacteroides (Phocaeicola) dorei or Bacteroides (Phocaeicola) vulgatus. This study was also interested in transcriptomic changes occurring in the Bacteroides partner as well. Biofilms were established by inoculating tissue culture–treated 24-well plates with 1 ml of Schaedler anaerobe broth supplemented with vitamin K (0.005 g/L) and L-cysteine (2 g/L L-cysteine), at a final inoculum density of OD600 = 0.05 per species. Cultures were incubated anaerobically for 24 h. Total RNA was extracted using TRIzol reagent, and library preparation and sequencing were performed by Genewiz.biostudies-arrayexpressSequencing - 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 Azenta/Genewiz.Nucleic Acid Extraction - Biofilms were resuspended in 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. The resuspensions were then 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. The samples were then quantified for both DNA and RNA using Qubit BR DNA and RNA kits (Thermo Fisher Scientific, USA).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 Azenta/Genewiz.Sample Collection - Single colonies were inoculated overnight into 10 mL Schaedler anaerobe (SAB) broth or agar (Oxoid, UK), routinely supplemented with 2 g/L L-cysteine (Sigma-Aldrich, USA) and 0.005 g/L vitamin K (referred as SAB+ broth). After approximately 14-15 h of growth, overnight cultures were normalised to 0.1 OD600 in pre-reduced SAB+ liquid media. For cocultures, 0.5 mL of each 0.1 OD600 species dilution were mixed, yielding a final concentration of 0.05 OD600 for each species within the coculture mix. For single species control biofilms, 0.5 mL 0.1 OD600 dilution was mixed with fresh pre-reduced SAB+ media. 1 mL aliquots of each dilution culture were added to pre-reduced 24-well tissue culture treated polystyrene plates and left to form biofilm until the desired timepiont(Falcon®, Corning, USA). Wells containing SAB+ alone were used as negative controls.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Fastq files were aligned using HISAT-2, and Libinorm was used to generate count files. Processed data uploaded here is the output of Libinorm ran in HT-seq compatible mode. Concatenated genomes or annotations files were used for coculture samples.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 RNAPhocaeicola vulgatusBacteroides-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 UnnikrishnanfalseBacteroides (Phocaeicola)-driven metabolic remodelling suppresses Clostridioides difficile toxin expression in mixed biofilm communitiesThe aim of this study was to investigate transcriptomic changes in Clostridioides difficile when cultured as monoculture biofilms or as co-culture biofilms with either Bacteroides (Phocaeicola) dorei or Bacteroides (Phocaeicola) vulgatus. This study was also interested in transcriptomic changes occurring in the Bacteroides partner as well. Biofilms were established by inoculating tissue culture–treated 24-well plates with 1 ml of Schaedler anaerobe broth supplemented with vitamin K (0.005 g/L) and L-cysteine (2 g/L L-cysteine), at a final inoculum density of OD600 = 0.05 per species. Cultures were incubated anaerobically for 24 h. Total RNA was extracted using TRIzol reagent, and library preparation and sequencing were performed by Genewiz.2026-09-23T00:00:00Z2026-09-23T01:00:46.415Z2025-09-30T22:47:25.49ZE-MTAB-15641ERP180839EFO_0002944EFO_0004170EFO_0009653EFO_0005518EFO_0003816EFO_000418410.1101/2025.09.03.674004