Naoya Yamashita

Assessment of safety of lactobacillus strains based on resistance to host innate defense mechanisms.

ABSTRACT Seven Lactobacillus strains belonging to four species were evaluated for pathogenicity as well as for in vitro sensitivity to the bactericidal mechanisms of macrophages in a rabbit infective endocarditis (IE) model. Two bacteremia-associated strains, L. rhamnosus PHLS A103/70 and L. casei PHLS A357/84, as well as the L. rhamnosus type strain and the probiotic L. rhamnosus strain ATCC 53103, showed moderate infectivity, and the virulence of the probiotic L. casei strain Shirota and type strains such as L. acidophilus ATCC 4356T and L. gasseri DSM 20243T in the model was negligible. The strains that showed pathogenic potential in the rabbit IE model (PHLS A357/84, PHLS A103/70, and ATCC 53103) were more resistant than strain Shirota to intracellular killing activity by mouse macrophages in vitro and also to bactericidal nitrogen intermediates, such as nitric oxide and NO2− ions. These results suggest that resistance to host innate defense systems, which would function at inflammatory lesions, should be considered in the safety assessment of Lactobacillus strains.

Detection of Verocytotoxin from Stool and Serological Testing of Patients with Diarrhea Caused by Escherichia coli 0157:H7

The detecion of verocytotoxin (VT) in stool and measurement of antibodies against VT and three antigens (unheated‐antigen, LPS, and flagellin) of Escherichia coli O157: H7 in the serum of patients with diarrhea were examined. Five of 14 inpatients during an outbreak had fecal VT2 in stool taken within 5 days of onset to hospitalization. Among these 5, 3 of them also had fecal VT‐producing E. coli (VTEC) serotype O157: H7, whereas the other 2 did not. In the passive hemagglutination (PHA) test with formalinized sheep red blood cells sensitized with theee VTEC O157: H7 antigens, 49 (74.2%) of 66 outbreak patients and 3 of 3 sporadic cases had antibodies against both or one of unheated‐antigen and LPS of E. coli O157, but none had antibody against flagellin. In addition, anti‐VT2 antibody was demonstrated in serum samples from 15 (94%) of 16 inpatients and 2 (4%) of 50 outpatients in an outbreak by a VT‐enzyme‐linked immunosorbent assay (VT‐ELISA). These results showed that serological assay particularly for antibodies against VT and unheated‐antigen or LPS of VTEC O157 may provide a useful tool for diagnosis of infection with VTEC O157.

Evaluation of serodiagnosis of toxoplasmosis by using the recombinant nucleoside triphosphate hydrolase isoforms expressed in Escherichia coli.

The nucleoside triphosphate hydrolase (NTPase) isoforms termed, NTPase-I and NTPase-II of Toxoplasma gondii, were expressed in Escherichia coli as inclusion bodies and purified under denaturing condition. Furthermore, NTPase-I was refolded as an active form and purified under non-denaturing condition. The purified NTPase isoforms, both denatured and refolded, were tested for their usefulness as antigens for the serodiagnosis of acute toxoplasmosis in immunocompetent humans. The test was conducted by using the recombinant NTPase isoforms and comparing the enzyme linked immunosorbent assay (ELISA) absorbances with the Sabin-Feldman dye test titer. Seventy-three sera from dye test-positive patients, and 30 sera from subjects with no T. gondii infection were examined. The total positive rates in dye test positive sera were: 82% (60/73) for denatured NTPase-I; 78% (57/73) for denatured NTPase-II; and 63% (46/73) for refolded NTPase-I. For all three antigen types of recombinant NTPase, the positive rates of sera of acute toxoplasmosis suspected patients were 93% (13/14). A moderate correlation between the ELISA absorbance using these antigens and the dye test titer was observed with the correlation coefficients, 0.583 (r2) for denatured NTPase-I, 0.472 (r2) for denatured NTPase-II, and 0.604 (r2) for refolded NTPase-I in the linear regression analysis. There was no significant difference observed in the antigenicity between refolded and denatured NTPase-I, nor between the isoforms.