Heterotrophic plate count (HPC) test has been employed to indicate the effectiveness of water treatment processes and the microbiological condition of the distribution system. In Japan, because the majority of HPC bacteria are supposed to be harmless and all tap water should maintain residual chlorine, there are few studies of the virulence of HPC bacteria. In this study, we examined HPC bacteria isolated from finished and tap water for hemolytic activity to determine their virulence potential. 34 of 39 colonies expressing hemolytic activity were identified by partial 16S rDNA sequencing, but some of their percent identity were relatively low. This may have been due to the mismatching of the primer pair with some strains, or these strains may be unidentified new species. A total of 30 of 34 isolates identified have been reported to be opportunistic pathogens or food poisoning bacteria. To control the growth of these opportunistic pathogens among HPC bacteria, appropriate water quality control must always be done and residual chlorine must be maintained in every tap for a safe water supply.

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The efficacy of gaseous chlorine dioxide (ClO₂) against feline calicivirus (FCV), a norovirus surrogate, in the dry and the wet states on a hard surface was evaluated. We demonstrated that low-concentration ClO₂ gas (mean 0.08 ppm, 0.22 μg/l) could inactivate FCV in the wet state with 0.5% fetal bovine serum (FBS) within 6 h in 45 to 55% relative humidity (RH) (>3 log₁₀ reductions) and FCV in the dry state with 2% FBS (percentage of FBS in the viral suspension) within 10 h in 75 to 85% RH (>3 log₁₀ reductions) at 20°C, respectively. Furthermore, a <0.3 ppm concentration of ClO₂ gas (mean 0.26 ppm, 0.73 μg/l) could inactivate (below the detection limit) FCV in the dry state with 5% FBS within 24 h in 75 to 85% RH at 20°C. In contrast, in 45 to 55% RH at 20°C, ClO₂ gas had little effect even when the FCV in the dry state was exposed to high-concentration ClO₂ (mean 8 ppm, 22.4 μg/l) for 24 h. These results suggest that humidity plays an important role in the inactivation by ClO₂ gas of FCV in the dry state. According to the International Chemical Safety Card, threshold limit values for ClO₂ gas are 0.1 ppm as an 8-h time-weighted average and 0.3 ppm as a 15 min short-term exposure limit. From these data, we propose that the treatment of wet areas of human activity such as kitchens, toilets, etc., with low-concentration ClO₂ gas would be useful for reducing the risk of infection by noroviruses (NV) without adverse effects. In addition, we believe that the application of a combination of a <0.3 ppm concentration of ClO₂ gas and a humidifier in places without human activity may make it possible to inactivate NV in the dry state on any surface within a contaminated room without serious adverse effects.

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Pseudomonas aeruginosa strains were isolated from 45 of 370 (12.2%) cockroaches captured in hospitals. By cockroach species, the bacterial strains were isolated from 39 of 181 (21.5%) Periplaneta fuliginosa and 6 of 183 (3.3%) Blattella germanica, showing a significant difference (p<0.01). Many P. aeruginosa-carrying cockroaches inhabited locker rooms (66.7%) and kitchens (17.8%). In terms of serotyping, many isolates were typed into groups A, G, and B. In drug sensitivity tests, strains showed the highest sensitivity to ciprofloxacin with an MIC₉₀ of 0.25 μg/ml, followed by 2 μg/ml meropenem, and 4 μg/ml ceftazidime, gentamicin, and ofloxacin. In contrast, many strains were resistant to cefotaxime and minocycline, accounting for 86.7% of all resistant strains. However, there was no multidrug-resistant P. aeruginosa strain, and all strains were negative for the metallo-β-lactamase gene (IMP-1 and VIM-2). These findings suggested that cockroach-derived P. aeruginosa may contaminate hospital environments, for which the control of disease-carrying insects in hospitals is important.

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One hundred and seven soil samples were collected from various places in Japan, and their bisphenol-A (BPA, 2,2-bis(4-Hydroxyphenyl)propane) degradative capacities were evaluated. Eighty-five soil samples possessed BPA degradative capacities, and 26 bacterial strains could be isolated as BPA-degrading bacterium. Sequence analysis of their 16S rRNA genes indicated that 22 isolates belonged to proteobacteria groups, and three of four Gram-positive bacterial strains, YA27, NO13, and NO15, were classified as Bacilli. All isolates except strain YA27 completely degraded 115 μg/mL BPA in L medium but strain YA27 degraded only 50 μg/mL BPA. Strain YA27 and three Sphingomonas sp. strains could also grow in basal salt media containing BPA as a sole carbon source (BSMB medium). In HPLC analyses, some isolates, including the three Sphingomonas strains, produced some BPA metabolites in their cultures although the others, including strain YA27, produced no detectable metabolite. Furthermore, the Pseudomonas strains SU1 and SU4 produced some BPA metabolites that were different from the metabolites detected in the degradation of BPA by the S. bisphenolicum strain AO1. These results suggested that all isolates could be applicable to the bioremediation of BPA-polluted soil and water. Furthermore, we suggest that Bacillus sp. YA27 and Pseudomonas SU1 and SU4 may exhibit novel BPA metabolism pathways that are distinct from that of S. bisphenolicum AO1.

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Candida antarctica lipase B (CALB) has been used to polymerize and degrade polyesters. We developed a convenient method for investigating the biodegradability of plastics that involves the use of CALB-displaying arming yeast. Polyurethane containing dulcitol units was prepared and used as the model material. Additionally, standard polyurethane with no dulcitol units was prepared by reacting 2,4-toluene diisocyanate with ethylene glycol. These polymers were incubated with CALB-displaying yeast cells. The polyurethane containing dulcitol was degraded, while the standard polyurethane was relatively unaffected. Arming yeast displaying appropriate enzymes can be used to investigate the biodegradability of synthetic plastics. It was also revealed that arming yeasts were applicable to evaluate the degradation of the film state of polyurethane.

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Before the present study, no fungi using anion surfactant as a nutrient had been identified, although some fungi were known to use nonion surfactant. Washing water collected from 63 washing machines was inoculated onto 0.1% LAS (Sodium dodecyl benzenesulfonate) anion surfactant media to identify fungi that can feed on anion-surfactant. Small dark colonies of fungi were found on several of the Petri-dishes from 12 days after inoculation. These were identified as Cladophialophora boppii and Exophiala spinifera using morphological features and rDNA data. A number of the isolates of C. boppii specifically were recognized as using anion surfactant as a nutrient. The growth characteristics of the two fungal species were examined on surfactant media of three kinds. Apart from anion surfactant, the fungi were also able to grow on nonion surfactant and on soap. The application of these fungi for environmental cleansing after detergent pollution is also discussed.

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As part of an epidemiological study on legionellosis, we attempted to isolate Legionella spp. from hot spring water samples, and were able to isolate Legionella micdadei from 3 (5.5%) of 55 samples. All of these isolates were able to grow within Acanthamoeba sp., suggesting that the isolates will be pathogens. We also confirmed that the K-2 strain from hot spring water grew in guinea pig monocytes. Sensitivity tests using 10 drugs showed that the isolates were most sensitive to imipenem, with the MIC₉₀ of 0.032 μg/ml, were least sensitive to minocycline, with the MIC₉₀ of 4 μg/ml, and were not sensitive to low amounts of other drugs.

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