The abscess forming abilities of "Streptococcus milleri" strains (Streptococcus constellatus, Streptococcus anginosus, and Streptococcus intermedius) isolated from dentoalveolar abscesses and the synergistic effect of Fusobacterium nucleatum co-inoculated with the isolates were examined on a mouse subcutaneous abscess model. Five days after inoculation, all S. milleri strains formed abscesses, which showed less pathological spread to surrounding connective tissues than those formed by Staphylococcus aureus 209P strain and were similar to those by F nucleatum ATCC25586. When each S. milleri strain and F nucleatum were co-inoculated, abscess sizes and each bacterial number recovered from abscesses increased in comparison to those treated by bacterial mono-inoculation of each S. milleri strain or F nucleatum alone. The strongest synergistic effect was observed in the combination of S. constellatus and F. nucleatum. In a time course experiment with this combination, the recovery of S. constellatus subsequently decreased after the decrement of F. nucleatum, and it appeared that the association with F nucleatum maintained the bacterial number of S. constellatus in the abscess. The cell-free supernatant of F nucleatum had a tendency to increase the abscess size caused by S. constellatus in this model. When S. constellatus was cultured with F. nucleatum culture supernatant in vitro, growth enhancement in the early phase was observed. Furthermore, the phagocytic killing of S. constellatus by human polymorphonuclear leukocytes (PMNs) was significantly suppressed and the PMN membranes appeared to be injured by addition of the F. nucleatum culture supernatant. These results suggest that the pathogenicity of S. milleri strains in odontogenic infections may be enhanced by the co-existence of F. nucleatum .

The aim of this study was to establish an identification method for the anginosus group within the genus Streptococcus by polymerase chain reaction (PCR). Using a primer pair based on the group-specific sequences of penicillin-binding protein 2B (pbp2b) gene, a 275-bp fragment was amplified from each species in the group but no size-matched products were obtained in other streptococci. Further identification in the species or subspecies level was possible by a multiplex PCR with primers for the 16S ribosomal RNA gene of Streptococcus anginosus, the hyaluronate lyase genes both of Streptococcus intermedius and Streptococcus constellatus subsp. constellatus, and the intermedilysin (ily) gene of S. intermedius. In the case ofStreptococcus constellatus subsp. pharyngis, the amplified fragment from the S. intermedius-type hyaluronate lyase gene was obtained, while that from the ily gene was not. These results also indicate that two different hyaluronate lyase genes are distributed among the anginosus group.

Hyaluronate lyase (HAase) genes of Streptococcus intermedius and Streptococcus constellatus subsp. constellatus were isolated. In S. constellatus subsp. constellatus, the deduced amino acid sequence of HAase was most similar to that of S. intermedius (68%), whereas the enzyme of S. intermedius was most similar to that of S. pneumoniae (72%). Upstream of the HAase gene on the opposite strands, an open reading frame of a putative glutathione peroxidase started in S. intermedius, and this arrangement was similar to that in S. pneumoniae but unlike that in S. constellatus subsp. constellatus. Cell lysates of Escherichia coli carrying each streptococcal gene showed HAase activity, demonstrating that each cloned gene actually coded for HAase.

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Hyaluronidase (HAase) activity was detected in both a human pus sample and the culture supernatant of the only bacterial isolate from the pus, Streptococcus intermedius, using a zymographic technique. The optimum pH range for HAase activity was similar for both samples. Although the bands showing the strongest HAase activity of these samples differed from each other with respect to molecular size, both samples were equally inhibited by an antiserum raised against HAase of S. intermedius. These results suggest that S. intermedius may produce HAase in vivo as well as in vitro, and that this enzyme and/or its fragments may play an important role in host tissue degradation.