Ayuko Takao

Abscess Forming Ability of Streptococcus milleri Group: Synergistic Effect with Fusobacterium nucleatum

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.

Sialidase of Streptococcus intermedius: a putative virulence factor modifying sugar chains

A sialidase gene of Streptococcus intermedius was cloned. It was most similar to nanA, a major sialidase gene in Streptococcus pneumoniae, and was expressed in Escherichia coli. Since the gene‐knockout S. intermedius strain lost detectable sialidase activity, the gene might code, either solely or mainly, the glycosidase in the bacterial genome. Polymerase chain reaction using the primers for the nanA homologue in S. intermedius (described as nanA below) showed that this sialidase gene was commonly distributed within the isolates of S. intermedius, but not found in the strains of other species among the anginosus group. In biofilm formation assay under cultivation with mucin, the nanA‐deleted S. intermedius maintained the amount of biofilm for 72 hr, while that of the parent strain decreased during incubation from 24 to 72 hr. Since sialidase activity in the parent strain increased during that time period, sialidase might contribute to the degradation of biofilm under sialic acid‐rich conditions. When S. intermedius was added into the HepG2 hepatoma culture, the calculated disassociation constant (Kd) of EDTA‐releasable bacterial adhesion to the cells was higher in the nanA‐deleted strain than in the parent. Furthermore, the rate constant, assuming endocytosis of the bacterium mediated by ASGP‐R in HepG2 cells, seemed to be increased by sialidase pretreatment of the bacterial cells before addition to the cell culture. According to the results, modification of sugar chains by sialidase on the bacterial surface and in the surrounding environment might influence both bacterial interaction and host–bacterial interaction in S. intermedius.

Hyaluronidase activity in human pus from which Streptococcus intermedius was isolated

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.

Cloning and expression of hyaluronate lyase genes of Streptococcus intermedius and Streptococcus constellatus subsp. Constellatus

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.

Rapid screening method for detecting highly pathogenic Streptococcus intermedius strains carrying a mutation in the lacR gene

Abstract Streptococcus intermedius is a member of the normal human commensal flora and secretes a human‐specific cytolysin intermedilysin (ILY) as a major virulence factor. Expression of ily is repressed by LacR and loss‐of‐function mutations of LacR are observed in many ILY high‐producing strains isolated from deep‐seated abscesses, suggesting that high ILY production is necessary for increased virulence. However, because ILY exhibits no &bgr;‐hemolysis on animal blood agar plates, differentiating ILY high‐ and low‐producing strains using conventional laboratory methods is not possible. Interestingly, S. intermedius also produces glycosidases, including MsgA and NanA, which exhibit N‐acetyl‐&bgr;‐d‐glucosaminidase and neuraminidase activities, respectively. Moreover, MsgA expression, but not NanA, is negatively regulated by LacR. Here we measured the activities of MsgA, NanA and ILY in strains isolated from clinical specimens and dental plaque to determine the correlation between these glycosidase activities and ILY hemolytic activity. Hemolytic activity showed a strong positive correlation with MsgA and a weak negative correlation with NanA activities. Therefore, we calculated the ratio of MsgA and NanA activity (M/N ratio). This value showed a stronger positive correlation (r = 0.81) with ILY hemolytic activity and many strains with high M/N ratios (>2) were ILY‐high producers with loss‐of‐function mutations in LacR. Figure. No Caption available.

Pharmacokinetics of S-1-Two way biochemical modulation for enhancing 5-FU delivery into tumor and reducing GI toxicities.

S-1 is an oral form mixture of tegafur (FT) which produces 5-FU, 5-chloro-2, 4-dihydroxypyridine (CDHP) which inhibits 5-FU degradation and oxonic acid (Oxo) which reduces 5-FU toxicities, in a molar ratio of 1 : 0.4 : 1. Pharmacokinetics of S-1 were studied using tumor-bearing animals. Bioavailahility : AUCpo/AUCiv ratios of FT, CDHP and 5-FU in rabbits were almost 100% with some individual differences, while that of Oxo was 10% or less. The ratio showed a tendency to be higher in fasting animals than in feeding animals, especially in the case of 5-FU (P bone marrow>spleen>kidney>GI tract>liver and plasma. However, it was noticeable that AUCtumor/AUCplasma ratio rather decreased as the escalation of S-1 dose. The reason would be that 5-FU increased non-linearly in the plasma and most normal tissues following the dose escalation of S-1. Based on these data, the optimum dosage of S-1 in humans should be established to maximize the anticancer effects and minimize the adverse reactions.