Maternal oral origin of Fusobacterium nucleatum in adverse pregnancy outcomes as determined using the 16S–23S rRNA gene intergenic transcribed spacer region

Fusobacterium nucleatum, a common Gram-negative anaerobe prevalent in the oral cavity, possesses the ability to colonize the amniotic cavity and the fetus. However, F. nucleatum may also be part of the vaginal microbiota from where it could reach the amniotic tissues. Due to the heterogeneity of F. nucleatum, consisting of five subspecies, analysis at the subspecies/strain level is desirable to determine its precise origin. The aims of this study were: (i) to evaluate the use of the 16S–23S rRNA gene intergenic transcribed spacer (ITS) region as a tool to differentiate subspecies of F. nucleatum, and (ii) to design a simplified technique based on the ITS to determine the origin of F. nucleatum strains associated with adverse pregnancy outcomes. Amplified fragments of the 16S–23S rRNA gene ITS region corresponding to the five subspecies of F. nucleatum were subjected to cloning and sequencing to characterize the different ribosomal operons of the subspecies. Distinctive length and sequence patterns with potential to be used for identification of the subspecies/strain were identified. These were used to evaluate the origin of F. nucleatum identified in neonatal gastric aspirates (swallowed amniotic fluid) by sequence comparisons with the respective oral and vaginal maternal samples. A simplified technique using a strain-specific primer in a more sensitive nested PCR was subsequently developed to analyse ten paired neonatal–maternal samples. Analysing the variable fragment of the ITS region allowed the identification of F. nucleatum subsp. polymorphum from an oral origin as potentially being involved in neonatal infections. Using a strain-specific primer, the F. nucleatum subsp. polymorphum strain was detected in both neonatal gastric aspirates and maternal oral samples in cases of preterm birth from mothers presenting with localized periodontal pockets. Interestingly, the same strain was not present in the vaginal sample of any case investigated. The 16S–23S rRNA gene ITS can be a useful tool to determine the origin of F. nucleatum. The results of this study strongly indicate that F. nucleatum subsp. polymorphum of oral origin could be involved with pregnancy complications.