Five ethyl (5-alkyl-2-amino-1,3-thiazol-4-yl) acetates (designated compounds 4a-e) incorporating octyl, decyl, dodecyl, tetradecyl, and hexadecyl alkyl chains, respectively, were prepared by reacting 4-alkyl-4-bromo-3-oxobutyric acid ethyl esters (3a-e) with thiourea in dried acetonitrile. Compounds 3a-e were synthesized by reacting alkylated ethyl acetoacetates with bromine. The newly synthesized compounds were characterized by mass spectrometry, NMR, and elemental analysis. Compounds 4a-c demonstrated good in vitro antiamoebic activity against Acanthamoeba polyphaga exposed to 10 mg L-1 for 6 h at 28 °C. Compound 4b showed the highest antiamoebic activity among the tested compounds, comparable to that of chlorhexidine dihydrochloride (CHX), decreasing the number of viable cells to below the detection limit of 1 cell mL-1. The activity of compounds 4a and 4c was similar to that of the commercial antifungal agent fluconazole (Flu). The cytotoxic and hemolytic activity of the compounds was assayed against human neonate dermal fibroblasts and sheep erythrocytes, respectively. Compounds 4a-c were less cytotoxic than Flu and CHX. Our results suggest that compound 4b, which is composed of a 2-amino-thiazole attached to a decyl group and an ethyl ester moiety, is a particularly safe and effective alternative amoebicidal agent.
Kudoa septempunctata is the causative agent of a foodborne disease associated with the consumption of raw Paralichthys olivaceus (olive flounder). Chickens were used to establish specific antibodies against K. septempunctata spores. A specific antiserum, CS#3, raised against sonicated spores, also recognized intact spores. The CS#3 antiserum showed high titers for sonicated and intact K. septempunctata spores and was suitable for both ELISA and immunohistochemical staining. Using homogenated raw olive flounder meat, the ELISA system detected more than 5.0×105 spores in 1 g of tissue, which was consistent with the number determined by microscopic examination. The preparation of rapid detection kits for K. septempunctata spores in P. olivaceus muscle tissue using immunochromatography with CS#3 antiserum should be useful for preventing the foodborne disease in the field.
In order to understand the microbial inactivation mechanism of high-pressure carbonation (HPC), we examined the changes in the activity of the respiratory chain and DNA damage in Escherichia coli cells. HPC was performed under 1-6 MPa at 30°C for 1 min. The increase in CO2 pressure decreased the number of viable cells of E. coli, intracellular ATP, and intracellular NADH, and increased the number of apurinic/apyrimidinic sites. These results indicate that HPC has a detrimental effect on the functioning of the respiratory chain in E. coli and induces DNA damage, which could result in the death of the bacterial cells.
Strain K-20T, a Gram-negative, nonmotile, nonspore-forming and strictly aerobic coccobacillus, which produces a pale pink pigment (R2A agar medium, 30℃, seven days) was isolated from a sample of biofilm obtained from a cooling tower in Tokyo, Japan. A phylogenetic analysis of the 16S rRNA partial gene sequences (1,439 bp) showed that the strain (accession number: AB297501) was related to Roseomonas frigidaquae CW67T and Roseomonas stagni HS-69T with 97.4% and 96.9% sequence similarity, respectively. Strain K-20T formed a distinct cluster with Roseomonas frigidaquae CW67T in the phylogenetic tree at a high bootstrap value (93%); however, distance was recognized between the strains. In addition, the DNA-DNA hybridization level between strain K-20T and Roseomonas frigidaquae JCM 15073T was 33%. The taxonomic data indicate that K-20T (=JCM 14634T =KCTC 32152T) should be classified in the genus Roseomonas as the type strain of a novel species, Roseomonas tokyonensis sp. nov.
The autofluorescence intensity of bacteria and fungal spores was quantified by fluorescence microscopy in order to obtain the information for evaluating fluorescence-based bioaerosol detectors and was comparable to that of some types of polystyrene microspheres (PSMs). Although the intensity for microbes was distributed across a wide range over an order of magnitude in gray scale, it was in the intensity range of certain PSMs. Furthermore, some of those bacteria and PSMs were aerosolized in a test chamber and the fluorescence intensity was measured with a bioaerosol detector. Although there was a slight difference in the order of intensity from the results obtained by fluorescence microscopy, the fluorescence-based bioaerosol detector showed the intensity was in a comparable range.
Some studies show the uptake of disinfectants on medical devices but no studies on their cytotoxicity have been reported. This study aimed to assess that cytotoxicity in a 3-dimensional culture system using HeLa cells grown in matrices composed of collagen. Plastic materials were soaked in the use solutions of the widely used high-level disinfectants, glutaraldehyde (GA), ortho-phthalaldehyde (OPA) and peracetic acid (PAA). After being rinsed, they were allowed to dry and were embedded into the cell medium to investigate the cytotoxicity of the residual disinfectants. Cytotoxicity was observed with the polyvinyl chloride, polyurethane and silicon tubes soaked in GA and OPA, indicating that both disinfectants were absorbed in the test pieces, whereas for PAA, none was observed. As for the polytetrafluoroethylene (PTFE) tubes, no disinfectant displayed cytotoxicity. GA and OPA are primary irritants, having a potential to cause anaphylaxis and other forms of allergic reactions. There should be consideration not only about the toxicity of the residual disinfectant from poor rinsing, but also about the toxicity that would result from the disinfectants that were absorbed and consequently released from the medical devices or materials.
Most critical instruments are not designed for heat sterilization and autoclaving. These items are usually treated with chemical agents such as peracetic acid(PAA), glutaraldehyde (GA) and ortho-phthalaldehyde (OPA). MTT assay is often used to evaluate the in vitro cytotoxicity of these chemical agents. In this study, disinfectants were allowed to come in direct contact with cells. Their cytotoxicity was evaluated based on cell viability and adhesive properties. The results obtained from the direct contact method were compared with those obtained from the conventional MTT assay wherein the disinfectants were added into a nutrient medium. It was found that the two methods yielded very different results, especially when aldehyde- and halogen-containing disinfectants were tested, and that toxicity may be underestimated in the MTT assay. Hence, it can be assumed that the direct contact assay is more accurate when evaluating the cytotoxicity of residual chemicals. It was also observed that the cytotoxicity of PAA was lower than that of GA and OPA.
The simplex real-time PCR assays based on the TaqMan probe and SYBR green I， which target cereulide synthetase genes (ces genes) were used for rapidly, reliably and sensitively identifying the emetic strains from among Bacillus cereus strains isolated from different sources. Only the emetic strains showed positive reactions to the real-time PCR assays, but all examined strains of diarrheal B. cereus, other Bacillus species and other gram positive and negative bacteria gave negative results. The final identification of emetic B. cereus was possible within 1 to 1.5 h in both simplex real-time PCR procedures. The detection limit of emetic strains in food was suggested to be 104-105 cfu/g. Both simplex real-time PCR assays were found to be rapid, sensitive and reliable diagnostic tools that complement the different cellular, immunological and chemical detection methods for cereulide- producing B. cereus, even if a relatively expensive device is required for the assays.