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Two Bacteroides fragilis neuraminidase-deficient mutants were used to study the role of neuraminidase activity in growth of B. fragilis in tissue culture monolayers (CHO cells) and in the in vivo rat granuloma pouch. The nanH structural gene for neuraminidase was cloned from B. fragilis TM4000 and was used to create two isogenic strains with chromosomal disruptions at the nanH gene. B. fragilis VRC404 contains an insertion flanked by disrupted copies of the nanH gene, and B. fragilis VRC426 contains a deletion of a significant portion of nanH coding sequences. The insertion mutant VRC404 is capable of reverting to nanH+. It grew as well as the wild type in CHO monolayers. However, between 48 and 72 h after infection, the bacterial population was enriched with nanH+ bacterial cells (10 to 20%). In the rat pouch 48 h after infection, more than 90% of the population sampled had become nanH+. The deletion mutant VRC426 showed a severe growth defect in the rat pouch model. In addition, VRC426 was efficiently outgrown by the wild type in competition experiments, even when the mutant was present at 10 times the number of wild-type cells at the time of infection. A common characteristic of both model systems is a drastic decrease in the free glucose concentration 16 to 24 h postinfection. We suggest that neuraminidase activity may be required for B. fragilis to grow to maximal levels in the tissue culture and rat pouch systems by making other carbon sources available after glucose levels are reduced.

A role for Bacteroides fragilis neuraminidase in bacterial growth in two model systems