Bacteroides species were anaerobically grown at 37☌ overnight in pre-reduced chopped-meat broth (Anaerobe systems, Morgan Hill, CA). Then 200 μl of the suspension was processed by intragastric gavage into each of the recipient GF mice. Reconstitution of GF mice with cecal contents and defined bacteria Cecal contents from single neonatal and 7-week old SPF mice were diluted into 50 ml (per one adult mouse) or 5 ml (per one 4, 12, 16-day old mouse) of PBS, and passed through a 70 μm cell strainer to eliminate clumps and debris under sterile conditions) under an atmosphere of 5% H2, 5% CO2 and 90% N2 in an anaerobic growth chamber (Coy Manufacturing, Grass Lake, MI). All animal studies were performed according to approved protocols by the University of Michigan Committee on the Use and Care of Animals.
The absence of microbiota was verified by microscopic analysis of stained cecal contents to detect unculturable contamination. GF mice were maintained in flexible film isolators and were checked weekly for germ-free status by aerobic and anaerobic culture. Wild-type mice on GF background were bred and maintained at the Germ-free Animal Core Facility of the University of Michigan. Subject type: Animal Subject species: Mus musculus (Factor headings shown in green) mb_sample_idįeces of GF or GF mice reconstituted with Esherichia coli (EC), Bacteroides acidifaciens (Bac), or Clostridia consortium (CL)Īnimals Specific-pathogen-free (SPF) C57BL/6 mice were originally purchased from Jackson Laboratories. These results identify the gut microbiota as a critical determinant of increased susceptibility to enteric infection during the neonatal period. The neonatal bacteria enhanced the ability of these protective Clostridiales to colonize the gut. Furthermore, intragastric administration of Clostridiales protected neonatal mice from pathogen infection. Conversely, depletion of Clostridiales abolished colonization resistance in adult mice. Administration of Clostridiales, but not Bacteroidales, restored colonization resistance and abrogated intestinal pathology upon pathogen challenge. The lack of colonization resistance was caused by the absence of Clostridiales in the neonatal microbiota. By colonizing adult germ-free mice with the cecal contents of neonatal and adult mice, we show that the neonatal microbiota is impaired in mediating colonization resistance against two major pathogens causing mortality in neonates. The high susceptibility of neonates to infections has been assumed to be due to immaturity of the immune system, but the mechanism remains unclear. Metabolome analysis of the cecal contents of GF mice and GF mice colonized with dominant gut microbes present in the ceca of neonatal and adult mice
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