GoKawiilMice
Wild-type mice on the C57BL/6 background and transgenic strains including µMT−/− (B6.129S2-Ighmtm1Cgn/J; 002288), Cd22−/− (C57BL/6-Cd22tm1Lam/J; 006940), C3−/− (B6.129S4-C3tm1Crr/J; 029661) and C1q−/− (B6(Cg)-C1qatm1d(EUCOMM)Wtsi/TennJ; 031675) were purchased from The Jackson Laboratory61,62,63,64 and maintained under specific pathogen-free housing conditions at Cincinnati Children’s Hospital. FcγRnull (FcRα null) mice with combined defects in FcγRI, FcγRII, FcγRIII, FcγRIV were provided by J. Ravetch46. All mice were housed under specific pathogen-free conditions in individually ventilated cages with ad libitum food (tekland 2919) and ultrafiltered UV-sterilized water under controlled environmental conditions including 22 °C, humidity 30–80% and 14 h–10 h light–dark daily cycles. Sex and age-matched groups of adult (6–8 weeks) mice were randomized for EcN colonization or no colonization control groups, and monitored for up to 150 days thereafter. For cross-fostering, pregnant mice were checked for delivery at least every 12 h, and pups were transferred to dams with or without EcN colonization after birth. Infected mice were checked at least every 12 h and euthanized when moribund for survival outcome experiments. All animal studies were performed under Cincinnati Children’s Hospital Research Foundation IACUC approved protocols.
Microbial colonization and infection
Features for E. coli strains Nissle 1917 (EcN), UTI89, RS218, SCB12, SCB29, SCB61, SCB34, SCB58, SCB37 and SCB60 are described in Extended Data Fig. 1d. EcN and other parental wild-type E. coli strains including EC958, MS7163 and RS218 (refs. 65,66,67; Extended Data Fig. 4a) and mutants with targeted defects in serotype-defining cell-surface structures (waaL, kpsD, fliC, rfaH or ompA) were generated by λ-Red-mediated homologous recombination as described37,38,68. Reference wild-type K. pneumoniae (43816), Yersinia enterocolitica (JB580v), Citrobacter koseri (BAA-895), Enterobacter cloacae (13047), Salmonella typhimurium (ST14028), L. monocytogenes (10403 s), S. aureus (USA200) and C. albicans (SC5314) have been described69,70,71,72,73,74,75. For oral inoculation, EcN was cultured overnight in brain–heart infusion (BHI) liquid medium, sub-cultured to early log-phase growth (OD 600 0.1; 37 °C; 200 rpm), washed and diluted in sterile saline and administered dropwise into the mouth of unanaesthetized adult mice (2 × 106 CFU in 50 µl). For infection, bacterial strains were cultured overnight in BHI liquid medium, sub-cultured to early log-phase growth (OD 600 0.1; 37 °C; 200 rpm), washed and diluted in sterile saline and administered intravenously via the lateral tail vein. Adult mice were infected with E. coli (5 × 106 CFU for bacterial burden quantification; 4 × 107 CFU for survival outcomes). Three-day-old neonatal mice were infected with E. coli (200 CFU in 50 µl, intraperitoneally or 5 × 106 CFU, oral gavage) as described6,76. Other bacteria including K. pneumoniae, L. monocytogenes and S. aureus were administered at the following dosages intravenously (1 × 105 CFU K. pneumoniae; 1 × 105 CFU L. monocytogenes; 7.5 × 106 CFU S. aureus in 200 µl) for infection in adult mice. For fungal infection, C. albicans was cultured overnight in yeast extract peptone adenine hemisulfate dextrose (YPAD) liquid medium, sub-cultured early log-phase growth (OD 600 0.1; 30 °C; 200 rpm), washed and diluted in sterile saline and administered intravenously to adult mice (1 × 106 CFU) as described77. The bacterial and fungal inoculum in each experiment was verified by spreading serial dilutions onto media agar plates. For enumerating the number of recoverable E. coli, K. pneumoniae, L. monocytogenes, S. aureus or C. albicans from colonized or infected mice, the faeces or tissue was dissected in sterile conditions and homogenized in saline containing 0.05% Triton X-100. Serial dilutions of the faeces or tissue homogenates were spread onto media agar plates, and the number of individual colonies enumerated after incubation at 37 °C for 24 h.
Serum collection and transfer
Blood collected from adult EcN-colonized or control mice via retroorbital or cheek bleeding, or from neonatal mice after decapitation, was allowed to clot at room temperature for 20 min and then centrifuged for serum collection. For adoptive transfer, sera from donor mice were pooled and transferred intraperitoneally to 8-week-old adult (100 µl) or 3-day-old neonatal (50 µl) recipient mice 4 h prior to infection. For IgG purification, frozen serum was thawed and purified using NAb Protein A Plus Spin Column (Thermo Fisher Scientific), quantified on the basis of optical density (NanoDrop), verified for purity by protein gel electrophoresis and Coomassie Blue staining, resuspended in sterile saline and administered intraperitoneally to 3-day-old neonatal recipient mice 1 h prior to infection.
Human specimens
Paired maternal and cord blood specimens after term pregnancy were collected through the IMPRINT cohort designed to investigate the effect of initial respiratory virus exposures on infant immunity78. This cohort began in October 2019 after receiving Institutional Review Board approvals from Cincinnati Children’s Hospital Medical Center and the University of Cincinnati Medical Center (IRB 2019-0629 and SITE00000489, respectively). Women were enroled in the third trimester of pregnancy, with collection of maternal sera and cord blood collected at delivery. Additional non-paired cord blood specimens from healthy donors were provided in a de-identified manner through Cincinnati Children’s Hospital Applied Gene and Cell Therapy Center, Cell Processing Facility. For newborn blood spots, 100 babies with neonatal sepsis were identified through International Classification of Diseases diagnostic codes ICD9-03842 and ICD10-P36.4 corresponding to ‘E. coli septicemia’ and ‘sepsis of the newborn due to E. coli’, respectively. ICD9-03842-based identification was limited to individuals ≤28 days old to identify neonatal infection (Fig. 4b). Three control babies without infection were individually matched for each case with identical sex, gestational age (±1 week) and birth timing (same month and year), except for two babies with E. coli sepsis born at 23 weeks of gestation, where only one matched control baby could be identified, were identified through the Michigan BioTrust for Health79. Three 6-mm circular punches from dried blood specimens for each baby with E. coli sepsis and corresponding control babies without infection were provided in a de-identified manner. All studies were performed under Cincinnati Children's Hospital Medical Center and Michigan Department of Health and Human Services IRB approved protocols.
Enzyme-linked immunosorbent assay
Antibodies against E. coli and other bacteria and fungi were titred using high-binding 96-well immunoassay plates (Corning, 9018) coated with microorganisms from log-phase (OD 600 0.5–0.7) growth, washed and diluted in saline and allowed to dry overnight under UV light. Thereafter, bacteria or fungi-coated plates were blocked with milk (3%), serum titred starting at a 1:10 dilution with 5 additional 1:5 serial dilutions and probed with biotin-conjugated secondary antibodies including: rat anti-mouse IgG (ThermoFisher 13-4013-85 (RRID AB_466650); 1:2,000 dilution), rat anti-mouse IgM (ThermoFisher 13-5890-82 (RRID AB_466761); 1:2,000 dilution), rat anti-mouse IgA (ThermoFisher 13-5994-82 (RRID AB_466863); 1:2,000 dilution), rat anti-mouse IgE (BD Pharmingen 553419 (RRID AB_394850); 1:2,000 dilution), rat anti-mouse IgG1 (BD Pharmingen 553441 (RRID AB_394861); 1:2,000 dilution), rat anti-mouse IgG2b (BD Pharmingen 553393 (RRID AB_394831); 1:2,000 dilution), rabbit anti-mouse IgG2c (ThermoFisher SA5-10235 (RRID AB_2810193); 1:2,000 dilution) and rat anti-mouse IgG3 (BD Pharmingen 553401; RRID AB_394838; 1:2,000 dilution).
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