The antibacterial activities of peppermint oil and its 53 constituents against Escherichia coil were examined in a preliminary screening test. Peppermint oil and 15 of its constituents had significant bactericidal activity against non-pathogenic E. coil and enterohemorrhagic E. coil O157: H7. Polyphenols from green tea and four catechins from green tea also had antibacterial activity aganist E. coil O157: H7 in liquid culture medium. Peppermint oil and three of its constituents, namely, menthol, menthone and neomenthol, killed the bacteria within one hour at concentrations above 400 μ g/ml in phosphate-buffered saline. Polyphenols from green tea showed bacteriostatic activity in the culture medium, and bactericidal activity in PBS. This latter effect only became evident after several hours. A synergistic effect was observed for peppermint oil and for menthol when combined with green-tea polyphenols. These results suggest that the antibacterial activities of peppermint oil and green-tea polyphenols might involve different mechanisms and combinations of peppermint oil or the active constituents of peppermint oil with green-tea polyphenols might be useful as natural antibacterial agents against E. coil 0157: H7.

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The secretion of extracellular ice-nucleating material (EIM) with ice nucleation proteins (INP) from an ice-nucleating bacterium, Erwinia uredovora KUIN-3, increased in proportion to the yeast extract concentration in the ice-nucleating medium. The level of this secretion could be exhibited in terms of the ice-nucleating temperature, T₅₀ (°C), using the ELIZA method with the anti-Ina A antiserum. The secretions and the production on the cell surface of all EIM containing the class A, B and C structures were activated by the addition of yeast extract. Furthermore, based on the examination involving the deletion of various medium components, it was found that lysine (Lys) was closely associated with the secretions of EIM in the class A and B structures. The addition of Lys could affect the polyamine content in the EIM, thereby changing the surface charge of EIM from 5.2 to 4.9. Also, the addition of cadaverine (CAD) could enhance the EIM secretion. Based on these results, the surface charge of the INP aggregate was shown to be essential for the translocation of INP and the secretion of EIM. We found that this secretion, especially the secretion of a class B structure, was inhibited by N, N'- dicyclohexylcarbodiimide (DCCD), which was an H⁺-ATPase inhibitor regardless of normal growth. We also found that this secretion required ATP and the positive charge on the EIM surface. This is the first report on the energy needed to secrete EIM into the culture broth and the possibility for controlling the production of the INP on the cell surface by the reduction of some compounds in the surroundings.

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N-Alkyl-2-benzylaminopyridinium iodides (2BAP-n) were synthesized from 2-benzylaminopyridine and n-alkyl iodide (alkyl group: hexyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl and octadecyl) at 80°C under 80MPa of static pressure, and their bactericidal characteristics were investigated with respect to their chemical structure and physicochemical properties. 2BAP-12 exhibited a wide and high range of bactericidal activity, and a higher activity than N-dodecylpyridinium iodide (P-12), which has only an alkyl group as a substituent on the pyridine ring, against all bacteria tested. The bactericidal activity of 2BAP-n against Escherichia coil K12 W3110 was affected by the carbon number of the alkyl chain. The plot of the activity of 2BAP-n against molecular hydrophobicity was found to be parabolic. From the result, it was suggested that the activity of 2BAP-n is dependent on the molecular hydrophobicity. Similarly, the activity had a linear dependence on the hydrophobicity of cell surfaces. The activity increased with an increase in the hydrophobicity, as well as in the case of general QACs. 2BAP-12 maintained high activity in the pH range from 5 to 8.5, though the activity was lower than that of P-12 in alkaline solution. It was thought that this exceptional bactericidal property is due to the effect of a large substituent, the benzylamino group, introduced onto the pyridine ring.

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Chromatographic analyses were carried out on bean curd refuse decomposed by Bacillus sp. HR6 to evaluate the decomposition process. The water-soluble fraction possessed strong absorption bands (251 - 259 nm). Gel filtration chromatography showed that one of the major peaks, at a molecular weight of 1170, disappeared and that two peaks with molecular weights of 1040 and 1310 were newly detected. In the ether- and chloroform-soluble fractions, a novel peak at the molecular weight of 8890 was observed after 24 h of decomposition, although we did not detect peaks from such higher molecular weights in the original sample. These results indicate that not only the decomposition of many kinds of water-soluble compounds but also the polymerization of hydrophobic ones were carried out in this process.

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The kinetics of microbial inactivation by microwave irradiation were studied from the viewpoint of thermal inactivation analysis. From the temperature history of a microbial suspension during irradiation, a thermal inactivation curve was predicted. The predicted curves were similar to experimentally produced curves by irradiation at various energy outputs, showing that the inactivation was predominantly due to the heat induced by irradiation. On the other hand, a small difference in the inactivation rate between the curves was observed at a high output. This implied the possible existence of nonthermal factors in irradiation lethality.

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Vibrio parahaemolyticus cells are known to lyse in distilled water on account of their osmotic fragility. When V. parahaemolyticus cells were subjected to osmotic shock, blebs were formed on cell envelopes, mainly at the septa of dividing cells and polar regions. Some of the blebs appeared to be detached from the cell surface to form vesicles. Our results indicate that surface blebs and vesicles formed by osmotic shock mainly involve the outer membrane. Although the cytoplasmic membrane itself is not released, its permeability barrier is disrupted.

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Antimicrobial actions of p-hydroxybenzoate alkyl esters (parabens) and two formalin derivatives, Quarternium 15 (Q15) and imidazolidinyl urea (IDU), which are commonly used in cosmetics and toiletries as preservatives or bactericides, were evaluated against Klebsiella pneumoniae using a technique of microbial calorimetry. With parabens, which are known to have bacteriostatic actions, increased concentrations caused changes in the growth thermograms of the microorganism characterized by a suppression in the slopes at the exponential growth phase. In contrast, with Q15 and IDU, which are known to have rather strong bactericidal effects the growth thermograms shifted toward a longer incubation time in proportion to the drug concentration while their shapes were unchanged. These two types of actions were compared in terms of two indexes: the changes in the growth rate constant and in the apparent growth retardation. In the case of bacteriostatic drugs such as parabens, antimicrobial actions resulted in the growth retardation due to the lowering of the growth rate constant. In contrast, bactericidal drugs such as formalin preservatives did not affect the growth rate constant and caused only an apparent retardation in growth. The observed difference in the change of growth thermogram patterns is discussed in terms of a difference in the two drug potency curves drawn on the basis of the two indexes.

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Quaternary ammonium salts (QAS), which are widely used as disinfectants, have the disadvantages of rusting and producing insoible salts in the presence of anionic surfactants. To deal with these drawbacks, we synthesized anti-rusting agent QAS by incorporating an alkyl phosphate anion to counter cationic surfactants. These new compounds have shown bactericidal effects and co-solubility. In this study, We evaluated the fungicidal activities of these new compounds, N-alkylN-2-hydroxyethyl-N, N-dimethylammonium alkyl phosphate (Pn-E analogues), N-alkylN-(2-hydroxy-3-phenoxy) propyl-N, N-dimethylammonium alkyl phosphate (Pn-PG1 analogues), as well as the effects of presently used disinfectants, [chlorhexidine digluconate (CHX), benzalkonium chloride (BAC), and digluconate (CHX), benzalkonium chloride (BAC), and alkyldiaminoethylglycine hydrochloride (ADE)], on pathogenic fungi, 3 strains ofCandida albicans, 2 strains ofCandida tropicalis, 1 strain ofCandida parapsilosis, 1 strain ofAspergillus niger, 2 strains ofAspergillus terreus, and 1 strain ofTrichophyton rubrum. Pn-E analogues and Pn-PG1 analogues at 0.1% concentration produced fungicidal effects onA niger only after 24 h but on other fungal strains after 1 h exposure. They showed the same fungicidal effects as those of BAC, CHX, and ADE on the 2 strains of C. tropicalis, and similar effects to those of BAC, CHX, and ADE inC. albicansdespite some differences among strains.

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Twenty laboratives participated in a collaborative study to evaluate methods for isolating Escherichia coil O157: H7 from radish sprouts. The collaborators attempted to isolate the organism from 25 g of radish sprouts inoculated with 28.3 CFU E. coli O157: H7, using enrichment and plating methods. E. coli O157: H7 could be isolated from most samples after being cultured at 42°C for 18 h in a modified EC broth supplemented with novobiocin but not after being cultured at 37°C for 6 h in tryptic soy broth.

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Ten strains of Bacillus subtilis from different sources were genotyped by pulsed-field gel electrophoresis (PFGE) after separate digestion with Acc II, BamH I or HindIII. Among the ten strains tested, five strains originated from patients and the food source incriminated in a B. subtilis food poisoning outbreak, four from foods and soil epidemiologically unrelated to the outbreak, and one was B. subtilis PCI 219. Although five strains from the outbreak displayed the same PFGE pattern after digestion of chromosomal DNA by three restrictionendonucleases, the other strains showed distinctly different patterns from each other. Thus, the PFGE analysis was suggested to provide an useful tool for the epidemiological survey on B. subtilis food poisoning outbreaks.

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A Bacillus subtilis sodA recombinant plasmid, pMW-sod, was introduced into a Sod-deficient strain E. coli IM303 (sodA, sodB). The activity of superoxide dismutase in the crude extract of E. coli IM303 bearing pMW-sod was markedly increased by the addition of an inducer, isopropyl-β-D-thiogalactopyranoside, in Mn²⁺-supplemented medium; in contrast, it was kept at the basal level in Fe²⁺-supplemented medium. However, the specific activity of B. subtilis SodA purified from this strain was extremely low, compared with that from B. subtilis. An atomic absorption analysis revealed that a relatively high content of Fe²⁺ was bound to this purified B. subtilis SodA. We further demonstrated that this purified B. subtilis SodA was activated by incubation with manganese salt. These results suggest that B. subtilis SodA was posttranslationally regulated by the manganese in vivo and in vitro.

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