A simple, rapid and reliable outline for identification of clostridia isolates from human infections was developed. It consists of a combination of API ZYM and API LRA Oxidase tests. The enzymatic activities were performed with strains sub-cultured onto carbohydrate-free medium (Columbia blood agar). Fifty-five strains of Clostridium difficile, C. bifermentans, C. sordellii, and C. perfringens from clinical specimens and eight reference standard strains representing different species of the same genus were analyzed. The accuracy of the new method was evaluated by comparison with the results obtained by DNA/DNA analysis.

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Capsular polysaccharides (CPSs) from six representative strains of Acetobacter diazotrophicus were isolated and fractionated by gel filtration and anion-exchange chromatography. Purified CPSs obtained in the non-adsorbed fraction of a DEAE-Sephadex A-25 column were qualitatively and quantitatively analyzed for sugar composition. Uronic acid and amino sugars were not detected in all purified CPSs. Basically the CPSs of A. diazotrophicus are composed of rhamnose, mannose, galactose and glucose. The presence of fucose was only observed in the CPS of strains PR2 and PAL3. Based on these results, the six strains of A. diazotrophicus could be divided into four groups according to the sugar content of their capsules: (i) fucose-containing capsules (PR2 and PAL3, localized in roots), (ii) mannose-rich capsule (PAL5, localized in root), (iii) capsules with a high ratio of hexose to rhamnose (PR4 and PR20, localized in stems) and (iv) capsules with a low ratio of hexose to rhamnose (PR14, localized in rhizosphere). For all CPSs, sodium dodecy sulfate-polyacrylamide gel electrophoresis showed diffuse bands of slow mobility in silver-stained gels. The different CPS migration patterns could not be correlated with sugar composition. The purified CPS of strain PAL3 was found to be immunogenic and immunochemically similar to the CPS of strain PR2. The serological specificity to CPS of strains PAL3 and PR2 correlated well with the presence of fucose, indicating that this deoxyhexose is immunodominant. These findings demonstrated the feasibility of preparing specific antibodies to fucose-containing CPS of A. diazotrophicus, indicating the possibility of utilization of this antiserum for future taxonomic studies or to select strains with chemically related capsular polysaccharides from their natural habitat.

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Artificial mutations of Gyrase A protein (GyrA) in Escherichia coli by site-directed mutagenesis were generated to analyze quinolone-resistant mechanisms. By genetic analysis of gyrA genes in a gyrA temperature sensitive (Ts) background, exchange of Ser at the NH₂-terminal 83rd position of GyrA to Trp, Leu, Phe, Tyr, Ala, Val, and Ile caused bacterial resistance to the quinolones, while exchange to Gly, Asn, Lys, Arg and Asp did not confer resistance. These results indicate that it is the most important for the 83rd amino acid residue to be hydrophobic in expressing the phenotype of resistance to the quinolones. These findings also suggest that the hydroxyl group of Ser would not play a major role in the quinolone-gyrase interaction and Ser83 would not interact directly with other amino acid residues.

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The effect of separate injections of two components of Clostridium perfringens iota toxin, designated I_a and I_b components, on the biological activities of the toxin was investigated. The intravenous injection of one component within 120min after the injection of the other component killed mice. The activity of iota toxin was abolished by anti-I_a or anti-I_b antiserum. On the other hand, when I_b component was intravenously administered to mice given anti-I_a antiserum within 120min after the intravenous injection of I_a component, the lethal activity was completely neutralized, but when I_a component was injected into mice that were given anti-I_b antiserum over 5min after the injection of I_b component, the activity was not neutralized. The separate injections of I_a and I_b components in skin of guinea pigs indicated dermonecrosis at the injection site of I_b components, but not at the site of I_a components. Furthermore, when one component was intradermally injected in guinea pigs and then the other intraperitoneally, the dermonecrotic activity of the toxin was observed at the intradermal injection site of I_b component, but not at that of I_a component. From the data, it appears that the lethal and dermonecrotic activities of iota toxin are initiated by the binding of I_b component to specific sites on tissues.

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Two monoclonal antibodies (MAbs) reacting with spirosins from Enterobacteriaceae were obtained in a course of screening MAbs to spirosin from Yersinia enterocolitica SYT-11-72 (YE72). The antibodies were designated MAbs-S44 and S50. They were IgG_2b and IgG_2a, respectively, both with κ light chains. On Western blotting after limited proteolysis of YE72 spirosin with Staphylococcus aureus V8 protease, they reacted markedly with peptide fragments of 27 and 35kDa, suggesting the presence of an antigenic determinant on the fragments. When supernatant cell lysate from Escherichia coli K12 was chromatographed on DEAE-cellulose and Sepharose CL-6B columns successively, a 96-kDa protein with alcohol dehydrogenase (ADH) activity was always associated with reactivity to MAb-S50. These findings combined with N-terminal amino acid sequences clearly indicate the identity of spirosin to ADH in E. coli.

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The human immune response to extracellular substances (ES) from Actinobacillus actinomycetemcomitans Y4 were analyzed. Twenty-nine periodontal patients with generalized severe alveolar bone loss and 13 healthy volunteers were examined for serum IgG antibody titers against ES. Among the patients, 17 had higher antibody titers than healthy individuals (high-titer patients) but the remainder (low-titer patients) did not. Two-dimensional electrophoresis (2DE) and Western blots demonstrated that two proteins (40 and 37kDa) and three smeared substances reacted with IgGs from high-titer patients, but not with IgGs from healthy volunteers. The low-molecular-mass smear at the acid side reacted with over 90% of all patients. This smear reacted with anti-A. actinomycetemcomitans lipopolysaccharide (LPS) monoclonal antibody which recognized LPS from each A. actinomycetemcomitans serotype. The high molecular mass smear at the acid side might be serotype-specific O-antigens of LPS. Another high molecular mass smear which located from the alkaline to the neutral side reacted with anti-serotype-b-specific capsular polysaccharide monoclonal antibody. Moreover, the 40- and 37-kDa proteins reacted with this anti-capsular polysaccharide antibody. This results suggested that 40-and 37-kDa proteins which reacted with 100% or 88% of high-titer patients might be glycoproteins linked with capsular polysaccharide.

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Cell-mediated immune response to Japanese encephalitis virus (JEV) and its purified envelope (E) protein was measured in 45 laboratory confirmed JE patients using a proliferation assay of peripheral blood mononuclear cells (PBMC). In parallel, JEV-specific IgM antibodies were measured by ELISA. No correlation was observed between the antibody response and results of the lymphocyte proliferation assay. Only 11 of the 42 patients positive in the antibody test were positive in the proliferation assay, and PBMC from 14/45 (31%) patients did not respond to either phytohemagglutinin and to JEV and/or its purified E protein antigen. No correlation was observed between the cell-mediated immune response and the final clinical outcome (fatality vs. recovery).

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We examined the effects of recombinant rat inteferon-gamma (IFN-γ) injections on the parasitologic, serologic, immunologic and histopathologic features of acute and chronic experimental Trypanosoma cruzi (T. cruzi) infections in“l”rats. Upon infection at weaning, two rat groups were allocated to receive a 20-day cycle of IFN-γ injections, 20, 000IU/rat each, which started at 1, and 7 days post-infection (pi). Treatment with IFN-γ, initiated at either 1 or 7 days pi, resulted in comparatively lower peak parasitemias (P<0.02) but in similar levels of anti-T. cruzi circulating antibodies and serum IFN-γ activities. The latter appeared significantly increased during acute infection whereas biologically active tumor necrosis factor was virtually undetectable in serum from infected rats regardless of whether they had been given IFN-γ or not. The prevalence of chronic focal myocarditis in IFN-γ-treated infected rats showed no differences with respect to the one recorded in control-infected counterparts. The inverse CD4/CD8 ratio of spleen and lymph node T cells that usually accompanies chronic infection was reversed by IFN-γ. Mononuclear cells carrying class II I-A and I-E molecules, that were found to have increased at both compartments, appeared also modified upon IFN-γ treatment with an overincrease of I-A-positive cells, and a normalization of I-E-bearing cells.

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A new selective plating medium for Rhodococcus equi containing ceftazidime (20mg/l) and novobiocin (25mg/l) on a Mueller-Hinton agar basis is described. It proved to be less inhibitory for R. equi than selective plating media devised earlier and grew only very few other nocardioform bacteria.

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By means of a polymerase chain reaction (PCR) method using subtype-specific primers for hepatitis C virus (HCV) subtypes 1a, 1b, 2a, 2b and 3a, the prevalence of each subtype among HCV isolates in Chiang Mai, Thailand, was determined. HCV-3a appeared to be the most common subtype in blood donors, and was also frequently found in patients with liver disease. HCV-1b, but not HCV-2a or -2b, was also commonly found in this area, while a considerable percentage of the total HCV isolates still remained unclassifiable by the above methods. Serotype analysis of the HCV isolates using C14-1 and C14-2 recombinant peptides revealed that HCV-3a was likely to carry an antigenic determinant(s) different from those of the major types 1 (HCV-1a and -1b) and 2 (HCV-2a and -2b).

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The natural killer (NK)-like cytotoxicity of murine intraepithelial lymphocytes in the small intestine (iIEL) and the participation of serine proteases in it were investigated. We monitored the cytotoxicity of iIEL with a sensitive cytotoxic assay using laser flow cytometry. iIEL exhibited NK-like cytotoxicity on YAC-1 target cells. Benzamidine, a serine protease inhibitor, inhibited significantly both Na-CBZ-L-lysine thiobenzyl ester (BLT)-specific serine protease activity and iIEL-mediated NK-like cytotoxicity. These results suggest that BLT-specific serine proteases may participate in NK-like cytotoxicity of murine iIEL.

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