Milk, Human

Human milk samples for HMO analysis were obtained by manual milk expression by the mother into a clean plastic container. The samples were kept cool until homogenization by the study team. They were then split into 1–2 mL portions and stored at −20°C. All aliquots were stored at −20°C at the study site until shipment on dry ice to the ETH Zurich, Switzerland. For the HMO composition analysis reported here, the HM samples were transported on dry ice to the glycoanalytical laboratory (glyXera GmbH, Magdeburg, Germany). The qualitative and quantitative HMO composition of each individual HM sample was determined with the glyXboxCE™ system (glyXera GmbH, Magdeburg, Germany) based on multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF).^{73 (link)} In accordance with the glyXera GmbH kit protocol (KIT-glyX-OS.P-APTS, glyXera GmbH, Magdeburg, Germany), the pure HM samples were diluted 1:100, spiked with an internal standard (IS) (oligosaccharide (OS) quantification standard solution, OS-A5-N-1 mL-01; part of the KIT-glyX-Quant-DP5, all from glyXera GmbH, Magdeburg, Germany) and treated with a denaturation solution. The free OS were labeled with 8-aminopyrene-1,3,6-trisulfonic acid (APTS), purified and determined with the glyXbox™ system. All measurements included the addition of a migration time alignment standard (glyXalign4; STD-glyXalign-4-S, glyXera GmbH) to the sample. Finally, glyXtoolGUI™ software (Beta v0.8.11, glyXera GmbH, Magdeburg, Germany) was used for the processing and analysis of the HMO Fingerprints data (normalized electropherograms). The limit of quantification (LOQ) was determined from the signal-to-noise ratio (SNR) of each HMO Fingerprint calculated as described by Ullsten et al.^{74 (link)} The LOQ was defined as an SNR of 10 and the limit of detection (LOD) was defined as an SNR of 3. The respective noise for each sample was determined after migration time alignment of the unsmoothed data in the late migration time range (approximation range = degree of polymerization (DP) 18< DP<20). Peaks with intensities below the LOQ but above the LOD were picked. All peaks ≥LOQ were considered and their IS-normalized peak areas were calculated (as percentages relative to the peak area of the IS [% IS] (= nPA)). All peaks ≥LOD but 75 (link) All HM samples were assigned to a maternal secretor and Lewis (Se/Le) phenotype (HM groups I–IV) based on the presence or absence of specific α1-2- and/or α1-4-fucosylated HMOs, as previously described.^{73 (link)} The assignment of maternal secretor status was based on the presence of 2’-fucosyllactose (2’-FL), difucosyllactose (DFL), and lacto-N-fucopentaose (LNFP) I, and the determination of Lewis status was based on the presence of LNFP II and lacto-N-difucohexaose (LNDFH) II. Differences in HMO abundance between maternal secretor status and HM types were assessed with Mann-Whitney tests or Kruskal-Wallis tests followed by post-hoc Dunn’s test, respectively, with adjustment for false discovery rate (FDR) by the Benjamini-Hochberg mechanism (FDR<0.05).

Fertilized eggs of a conventional flock of Coturnix japonica were obtained from the Poultry Experimental Facility (UE1295 PEAT) of INRAE (Nouzilly, France). They were incubated and aseptically transferred into a sterile hatching isolator two days before the expected hatching day as previously described.^{50 (link)} Six days after hatching, the young quails were transferred from the hatching isolator to experimental isolators and inoculated with a freshly prepared culture of a WT Clostridium strain (n = 13–15 quails) or of its KO strain (n = 12–15 quails). The WT and KO groups were housed in separate isolators. Inoculation was achieved by orally administering 0.2 mL of the bacterial culture to each quail. Bacterial sterility was checked before inoculation. The bacterial establishment was checked weekly using freshly collected droppings that were observed under an optical microscope and grown in appropriate culture media. Two weeks after bacterial inoculation (quail age: 3 weeks), the starter feed was gradually replaced within 4 days by a grower semisynthetic feed supplemented with 7% lactose (wt/wt) to mimic the proportion of lactose in human milk.^{51 (link)} Diets were manufactured by SAFE® diets (Augy, France) and sterilized by γ-irradiation at 45 kGy. The quails received diets and autoclaved drinking water ad libidum. Six to eight weeks after inoculation, a sufficient time for the development of NEC-like lesions,^{20 (link)} quails aged 7–9 weeks were deeply anesthetized with isoflurane, weighed, and sacrificed by decapitation. The ceca were removed for macroscopic examination and scoring of the lesions (Table S2). The cecal contents were collected and conserved at −80°C until bacterial enumeration and SCFA analysis; however, as the cecal content volume was usually not sufficient to carry out both analyses, some samples were dedicated to bacterial enumeration and the others to SCFA analysis. Empty ceca were weighed to quantify the wall thickening and cecal walls, chosen to represent the variety of macroscopic lesion scores, were fixed in 4% paraformaldehyde, stored at 20°C, and transferred to the APEX histology facility (UMR703 INRAE-Oniris, Nantes, France) for histological analyses. After embedding in paraffin wax, 5-µm-thick transversal ceca sections were stained using the hematoxylin–eosin–safranin staining method. All histological observations were performed blinded by a certified veterinary pathologist. Using low magnification, the mucosal thickness was measured five times per animal cecum on randomly scattered points (intra-assay variation calculated after five independent measurements on the same sample was 7.7%).
Animal experiments were organized as cohorts with simultaneously comparing the WT and KO groups of a given Clostridium species, namely C. butyricum or C. neonatale. Experiments were performed in the Anaxem germ-free animal facility (license number: B78-322-6; INRAE, Micalis Institute, Jouy-en-Josas, France). Procedures conformed to the European guidelines for the care and use of laboratory animals and were approved by the Animal Ethics Committee of the INRAE Research Center, Jouy-en-Josas (approval reference: APAFIS#2540-2015120315579065 v2).