The present study was carried out to determine the surface hydrophobicity of group A streptococcal strains responsible for rheumatic fever (RF), “rheumatogenic” strains (RG strains) and strains causing glomerulonephritis, “nephritogenic” strains (NG strains) in relation to their adhesion to human pharyngeal cells. Scanning electronmicroscopic (SEM) studies were carried out to the difference, if any, in the adherence of group A streptococci (M type 5) to pharyngeal and buccal cells (PEC and BEC). By employing two techniques for hydrophobicity determination, salt aggregation titre (SAT) and n-hexadecane binding technique, it was observed that RG strains (M5, M1 and M6) were more hydrophobic than NG strain, M49. However, NG strain M12 was almost equally as hydrophobic as RG strains. The adherence of RG strains, except M1 and M24, to PEC was greater in number than that of NG strains. Although M1 strain was hydrophobic, its adherence to PEC was less. Pepsin and trypsin treatment with streptococci reduced the hydrophobicity and adherence of RG and NG strains to PEC. SEM studies revealed firmly adhered indigenous bacteria on PEC and BEC. Streptococci (M5) adhered more to PEC than to BEC. SEM studies also showed that PEC had a peculiar ultrastructural surface feature to which streptococci adhered. These findings suggest that streptococcal hydrophobicity alone does not determine their adhesion to PEC. The surface nature of PEC might be a characteristic feature of the epithelial cells that allows streptococci to adhere and colonize or it might be a consequence of streptococcal adhesion.

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The quantitative, semi-automated assay described here is an alternative characterization method allowing for highly sensitive and specific detection of bifidobacterial enzymes. Twenty strains of Bifidobacterium longum, including the type strain ATCC 15707, and type strains of 15 other Bifidobacterium species were enzymatically characterized using 20 4-methylumbelliferyl conjugated substrates. Enzyme activities were determined by directly measuring the intensity of fluorescence derived from 4-methylumbelliferone, a fluorescent metabolic by-product. For this method, a Titertek^® Fluoroskan II fluorometer was used. Enzymes included glycosidases, an esterase, phosphatase, sulphatase, and neuraminidase. B. longum showed strong activity (>1, 000 absolute fluorescence units, afu) for α-L-Arabinopyranosidase and α-L-Arabinofuranosidase, β-D-Fucosidase, α- and β-D-Galactosidase, α-D-Glucosidase, and α-D-Mannosidase. No activity (≤50 afu) was observed for β-D-Cellobiosidase, α- and β-L-Fucosidase, β-D-Glucuronidase, β-D-Mannosidase, Neuraminidase and Sulphatase. Enzymatic activity profiles in other bifidobacteria were different according to the species. This assay is simple and rapid (6hr). Special cultural requirements are unnecessary. Results are objective and quantitative. This assay may be a useful tool for bifidobacterial taxonomy.

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Vibrio mimicus, a causative agent of gastroenteritis, has also been reported to attribute to extraintestinal infections. Recently we have purified a metalloprotease produced by the pathogen: however, the role of the protease in V. mimicus infection has not been documented. The V. mimicus protease (VMP) was found to enhance vascular permeability and form edema when injected into the dorsal skin of guinea pig and rat. The permeability enhancement by VMP was observed in a dose-dependent manner in both guinea pig and rat skin. In guinea pig, an inhibitor of the angiotensin-converting enzyme was found to augment the permeability enhancement reaction. The permeability enhancement was significantly blocked by soybean trypsin inhibitor (SBTI), an inhibitor of plasma kallikrein reaction. In vitro conversion of plasma prekallikrein to kallikrein by VMP was also noted. In rat skin, the permeability enhancement reaction was not blocked by antihistamine or SBTI. However, the reaction was partially blocked when a mixture of antihistamine and SBTI was administered with VMP. It is apparent from the study that in guinea pig skin, VMP enhances vascular permeability through activation of plasma kallikrein-kinin system which generates bradykinin, whereas in addition to the activation of plasma kallikrein-kinin cascade in the case of rat, stimulation of histamine release from mast cells and other unknown mechanism seem to be also a cause of the permeability enhancement reaction. These results suggest that VMP may play a role in extraintestinal infections with edema caused by the pathogen.

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The antibacterial activity of cefpodoxime against Branhamella catarrhalis was studied. All of the 65 clinical isolates tested were inhibited at and below 1.56 μg/ml, both at 10⁷ and at 10⁵ CFUs. The following was further studied on B. catarrhalis N-5 which showed average susceptibility to each drug examined. Bactericidal activity was observed at and above the MIC. Scanning and transmission electron microscopy revealed morphological changes, such as cellular swelling, bleb formation, inhibition of septum formation, and lysis, of the cells exposed to cefpodoxime at concentrations around the MIC. Cefpodoxime was poorly hydrolyzed by the β-lactamase and it showed affinity for two penicillin-binding proteins that had approximate molecular weights of 83 and 74 kilodaltons, with I₅₀ values of 3.7 and 2.1μg/ml, respectively.

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Two bacteriophages named φVP253 and φVP143 isolated after ultraviolet induction from lysogenic strains of Vibrio parahaemolyticus have been shown to be generalized transducing phages. So far, seven different auxotrophic markers of a V. parahaemolyticus strain could be transduced at the frequencies ranging from 2.2× 10^-7 to 7.5×10^-5 per infected cell at the m.o.i. of approximately 1.0. The phage φVP143, but not φVP253, lysed 20 of the 28 strains of V. alginolyticus and the occurrence of generalized transduction by this phage in this Vibrio species has been confirmed. Molecular size of the genomes of both phages were estimated to be approximately 48kb as judged from electrophoretic mobilities of the DNAs digested with HindIII endonuclease. The results and similarity of the two phages in morphology and other properties suggest very close relatedness of the phages.

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Yersinia enterocolitica is capable of growing in a broad range of temperatures from 4 to 45C. How this organism alters its membrane lipids in response to the change of growth temperature is very interesting. The fatty acids of membrane lipids of cells cultured at 5, 15, 25 and 37C were analyzed and the physical states of these membrane lipids were characterized. The major phospholipids of this bacterium were phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, lysophosphatidylglycerol and lysophosphatidylethanolamine. No significant difference in phospholipid composition in response to culture temperatures was observed. It was reported in our previous paper that the major fatty acids of membrane phospholipids of Y. enterocolitica were C_15:0, C_16:0, C_16:1, cyclopropane C_17:0 and C_18:0. Some differences in the fatty acid composition were, however, observed with the change of culture temperature. When the culture temperature was raised, the saturated and cyclopropane fatty acids substantially increased and the unsaturated ones decreased. A reverse phenomenon was observed when culture temperature was lowered. From the viewpoints of membrane physical state, adaptational changes were analyzed using a nylon microcapsule method. Phase transition in membrane lipids of cells grown at each culture temperature took place in the range of about 5C below and about 10C above the culture temperature. It is, therefore, considered that Y. enterocolitica maintains its membrane rigidity and fluidity in response to growth temperature by changing the membrane fatty acid composition.

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The germination of spores of a neurotoxigenic Clostridium butyricum strain (BL 6340), which was isolated from infant botulism in Italy, and that of a nontoxigenic C. butyricum type strain (NCIB 7423) were studied. The spores of BL 6340 strain were killed at 80C for 10min, and required the mixture of L-alanine, L-lactate, glucose and bicarbonate for their optimal germination. These characteristics are the same as those of Clostridium botulinum type E strain, but different from those of NCIB 7423 strain. In a hybridization test, however, the labeled DNAs extracted from NCIB 7423 strain highly (98%) hybridized to the DNAs of the BL 6340 strain, but little (45%) to the DNAs of C. botulinum type E strain. The biochemical properties of the BL 6340 and NCIB 7423 strains were identical, but different from those of C. botulinum type E. These data confirmed that the BL 6340 strain belongs to C. butyricum species, but that only its characteristics of toxin production, its minimum requirements for germination, and the behavior of its spores to heat treatment are the same as those of C. botulinum type E. When conventionally raised suckling mice were injected with 5×10⁷ spores of BL 6340 strain intra- or orogastrically, botulism was not observed. However, 8- to 13-day-old mice had type E botulinum toxin in the large intestine 3 days after introduction of its spores.

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In FL cells, interferon (IFN)-induced dsRNA-dependent protein kinase (PK-I) was found to be present in a form complexed with a potent inhibitor of its dsRNA-dependent activation. The inhibitor was readily dissociated from PK-I by DEAE-cellulose chromatography to yield a dsRNA-responsive PK-I. The inhibitor was also dissociated easily from PK-I by gel filtration through Sephacryl S-200. The apparent molecular mass of the inhibitor as estimated by gel filtration was more than 160 kilodaltons. Activity of the inhibitor was decreased on IFN treatment for 8.5hr or on Sindbis virus infection with concomitant increase in the amount of dsRNA-activatable form of PK-I. This result implies that the inhibitor may be one of the regulatory factors of cellular PK-I activity. Longer IFN treatment (24hr) led to recovery of the inhibitor activity, but it was overridden by an extensive net synthesis of the PK-I protein.

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Nylon wool-passed bone marrow (NW-BM) cells treated with anti-Thy.1 monoclonal antibody and complement were added to a mixed lymphocyte culture which contained a limiting number of lymph node cells, as responder cells, and a sufficient number of mitomycin-c-treated allogeneic spleen cells as stimulator cells. NW-BM cells of the same MHC haplotype as responder cells enhanced the generation of allo-specific cytotoxic T lymphocytes (CTL) not only at a relatively high dose (3×10³ cells/well) of responder cells, but also at an extremely dilute dose (1×10³ cells/well). NW-BM cells which had a third-party MHC haplotype, a haplotype different from both responder and stimulator cells, also enhanced the generation of CTL at relatively high doses, but not at low doses, of responder cells. NW-BM cells which had MHC haplotypes identical with those of responder cells induced CTL from helper T cell-depleted responder cells, but NW-BM cells which had the third-party haplotype did not. These results showed that the enhancing effects of NW-BM cells of the same MHC haplotype as responder cells might be due to a specific helper effect and the enhancing effect of NW-BM cells of the third-party haplotype might be due to a nonspecific filler effect, which only conditioned the cultured cells. It was also found that, to exhibit the helper effect, NW-BM cells had to possess MHC class II, but not MHC class I, molecules in common with CTL precursors. This study showed that in the induction of CTL, prethymic NW-BM cells had a capability comparable to that of mature helper T cells.

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We employed a Pseudomonas aeruginosa mouse pneumonia model to evaluate the ability of a murine monoclonal antibody (MAb) specific for the O-side chain of P. aeruginosa Fisher Immunotype-1 lipopolysaccharide (LPS) to achieve and sustain therapeutic levels in plasma and lung tissue, reduce bacterial populations in the lung, and prevent pneumonia-associated mortality. An IgG3 MAb (Y1-5A4) administered to mice i.v. over a dose range of 125-1, 000μg/mouse produced plasma and lung tissue levels at 2hr of 61-507μg/ml and 4.3-150μg/g, respectively. The 1, 000μg MAb dose reduced bacterial counts in lung tissue (log₁₀ cfu/g±S.D.) and blood (log₁₀ cfu/ml±S.D.) 20hr post-treatment (18hr post-challenge) from 10.00± 0.66 to 7.66±0.91 (P<0.01) and from 4.39±0.81 to <3.0, respectively. Administration of MAb to mice in doses of 125-500μg 2hr prior to a 3×50% lethal bacterial challenge produced significant protection against death, with a calculated 50% protective dose of 167μg. Protection was noted following administration of 1, 000μg of MAb up to 6hr after bacterial challenge (P<0.05, compared with untreated control). Histological examination of lung tissue from infected mice revealed less acute inflammation, necrosis, and hemorrhage in MAb-treated compared with untreated control animals and greater localization of Pseudomonas antigen within the phagocytic cells in alveolar space. These findings document the in vivo therapeutic efficacy of an LPS-specific IgG MAb in a murine model of acute P. aeruginosa pneumonia, based in part upon the achievability of effective MAb concentrations in plasma and lung tissue.

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Intraperitoneal injection of adsorbed diphtheria-purified pertussis-tetanus combined vaccine (DPPT) often causes an ascites-accumulating (A-A) reaction in guinea pigs. Those vaccines which caused A-A reaction increase the vascular permeability (VP) of the skin tissue at the intracutaneous injection sites. A total of 23 lots of DPPT were assayed for A-A and VP-increasing activities. A positive correlation between the volume of ascites accumulated and the intensity of VP was significant. Other kinds of aluminum-adsorbed (Al-Ad) vaccines such as Al-Ad tetanus and Al-Ad diphtheria-tetanus combined and Al-Ad hepatitis B vaccines induced little or no A-A reaction. These results were discussed from the viewpoint of quality control in the process of the production of DPPT.

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