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Enhancement of Bacteroides intermedius growth by Fusobacterium necrophorum
Abstract:
Previous work from this laboratory has demonstrated the persistence of Bacteroides intermedius in the livers of mice receiving an intraperitoneal inoculum of B. intermedius and Fusobacterium necrophorum. This study was undertaken to determine whether F. necrophorum enhanced the in vitro growth of B. intermedius. Tryptose phosphate broth did not support the growth of B. intermedius alone, but the bacterium did survive in a tryptose phosphate broth culture of F. necrophorum. B. intermedius cultured in F. necrophorum-conditioned tryptose phosphate broth grew impressively, reaching maximal absorbance at 24 h after inoculation. The growth of B. intermedius in F. necrophorum-conditioned tryptose phosphate broth was proportional to the amount of conditioned medium present. The B. intermedius growth-stimulating factor was detectable in conditioned medium 8 h after inoculation with F. necrophorum and could be detected throughout the 96-h incubation period. Growth-factor-active fractions eluted from a Sephadex G-100 column did not absorb at 280 nm and were retained on the column until 4 column volumes were eluted. The growth factor was nondialyzable and stable to boiling, lyophilization, extraction with hot aqueous phenol, and trypsin digestion. The factor was inactivated by exposure to pH 2.0 in the pepsin digestion protocol. Significant amounts of hexose, methyl pentose, and 2-keto-3-deoxyoctonate were detected in pooled growth-factor-active fractions eluted from the Sephadex column. This pool was also active in the Limulus lysate endotoxin assay. These results suggest that the B. intermedius growth-stimulating factor produced by F. necrophorum is a lipopolysaccharide.
Insights
Fusobacterium necrophorum produces a growth factor that significantly enhances Bacteroides intermedius growth in vitro. This lipopolysaccharide factor is stable and detectable in conditioned media, aiding B. intermedius survival and proliferation.
Area of Science:
- Microbiology
- Bacterial Interactions
- Host-Pathogen Dynamics
Background:
- Previous studies showed Bacteroides intermedius persists in mouse livers when co-inoculated with Fusobacterium necrophorum.
- The mechanism by which F. necrophorum influences B. intermedius survival and growth remained unclear.
Purpose of the Study:
- To investigate whether F. necrophorum enhances the in vitro growth of B. intermedius.
- To characterize the nature of the growth-stimulating factor produced by F. necrophorum.
Main Methods:
- Co-culturing B. intermedius and F. necrophorum in tryptose phosphate broth.
- Analyzing bacterial growth using absorbance measurements.
- Characterizing the growth factor using gel filtration (Sephadex G-100), stability assays (pH, heat, enzymatic digestion), and biochemical analysis (hexose, methyl pentose, KDO).
- Assessing endotoxin activity using the Limulus lysate assay.
Main Results:
- F. necrophorum-conditioned medium significantly promoted B. intermedius growth, unlike unconditioned medium.
- The growth-promoting activity was dose-dependent on the conditioned medium concentration.
- A stable, nondialyzable growth factor was detected in conditioned medium, present from 8 to 96 hours.
- Biochemical analysis revealed the presence of lipopolysaccharide components (hexose, methyl pentose, KDO) in active fractions.
- Active fractions also showed positive results in the Limulus lysate endotoxin assay.
Conclusions:
- Fusobacterium necrophorum produces a lipopolysaccharide (LPS) growth factor that stimulates Bacteroides intermedius proliferation in vitro.
- This LPS factor likely contributes to the in vivo persistence of B. intermedius observed in co-infection models.
- Understanding this interaction is crucial for deciphering polymicrobial infections involving these bacteria.