Effects of disinfectants against norovirus virus-like particles predict norovirus inactivation.

Human noroviruses (NoVs) are a major cause of epidemic and sporadic acute gastroenteritis worldwide. Public and personal hygiene is one of the most important countermeasures for preventing spread of NoV infection. However, no a practicable cell culture system for NoV had been developed, initial tests of the virucidal effectiveness of anti-NoV disinfectants and sanitizers have been performed using surrogate viruses. In this study, NoV virus-like particles (VLPs) were used as a new surrogate for NoVs and a method for evaluating NoV inactivation using them developed. This method is based on morphological changes in VLPs after treatment with sodium hypochlorite. VLP specimens were found to become deformed and degraded in a concentration-dependent manner. Based on these results, the effects of sodium hypochlorite on VLPs were classified into four phases according to morphological changes and number of particles. Using the criteria thus established, the efficacy of ethanol, carbonates and alkali solutions against VLPs was evaluated. Deformation and aggregation of VLPs were observed after treatment with these disinfectants under specific conditions. To determine the degradation mechanism(s), VLPs were examined by SDS-PAGE and immunoblotting after treatment with sodium hypochlorite and ethanol. The band corresponding to the major capsid protein, VP1, was not detected after treatment with sodium hypochlorite at concentrations greater than 500 ppm, but remained after treatment with ethanol. These results suggest that VLPs have excellent potential as a surrogate marker for NoVs and can be used in initial virucidal effectiveness tests to determine the mechanism(s) of chemical agents on NoVs.

Stress tolerance of Methylobacterium biofilms in bathrooms.

A comprehensive survey of microbial flora within pink biofilms in bathrooms was performed. Pink biofilms develop relatively rapidly in bathrooms, can be difficult to remove, and are quick to recur. Bacterium-sized cells were found to be predominant in 42 pink biofilms in Japan using a scanning electron microscope. Methylobacterium strains were detected from all samples in bathrooms by an isolation method. To explain this predominance, 14 biofilm samples were analyzed by fluorescence in situ hybridization. Methylobacterium was indicated to be the major genus in all biofilms. The isolated Methylobacterium survived after contact with 1.0% cleaning agents, including benzalkonium chloride for 24 h. Their tolerance did not differ under biofilm-like conditions on fiber reinforced plastics (FRP), a general material of bath tubs, floors, and walls. Also, the strains exhibited higher tolerance to desiccation than other isolated species on FRP. Some Methylobacterium survived and exhibited potential to grow after four weeks of desiccation without any nutrients. These specific characteristics could be a cause of their predominance in bathrooms, an environment with rapid flowing water, drying, low nutrients, and occasional exposure to cleaning agents.

Method for identifying heat-resistant fungi of the genus Neosartorya.

Species of the genus Neosartorya are heat-resistant fungi that cause the spoilage of heat-processed acidic foods due to the formation of heat-resistant ascospores, and they produce mycotoxins, such as fumitremorgins and gliotoxin. Their anamorphs are phylogenetically and morphologically very close to Aspergillus fumigatus, which has never been reported as a spoilage agent in heat-processed food products. Therefore it is important to discriminate between the species of Neosartorya and A. fumigatus in the food industry. In the present study, we examined ß-tubulin and calmodulin genes to identify Neosartorya and A. fumigatus at the species level and found a region for specifically detecting these species. We succeeded in developing the PCR method of differentiating and identifying Neosartorya and A. fumigatus using specific primer sets. Moreover, we developed specific primer sets to identify Neosartorya species, N. fischeri, N. glabra, N. hiratsukae, N. pseudofischeri, and N. spinosa-complex, which are important in food spoilage; these fungi vary in heat resistance and productivity of mycotoxins, depending on the species. PCR using these primer sets did not detect other fungi involved in food spoilage and environmental contamination. These identification methods are rapid and simple with extremely high specificity.

Moraxella species are primarily responsible for generating malodor in laundry.

Many people in Japan often detect an unpleasant odor generated from laundry that is hung to dry indoors or when using their already-dried laundry. Such an odor is often described as a "wet-and-dirty-dustcloth-like malodor" or an "acidic or sweaty odor." In this study, we isolated the major microorganisms associated with such a malodor, the major component of which has been identified as 4-methyl-3-hexenoic acid (4M3H). The isolates were identified as Moraxella osloensis by morphological observation and biochemical and phylogenetic tree analyses. M. osloensis has the potential to generate 4M3H in laundry. The bacterium is known to cause opportunistic infections but has never been known to generate a malodor in clothes. We found that M. osloensis exists at a high frequency in various living environments, particularly in laundry in Japan. The bacterium showed a high tolerance to desiccation and UV light irradiation, providing one of the possible reasons why they survive in laundry during and even after drying.

[Evaluation of the immunochromatography kit for detection of emetic-toxin producing Bacillus cereus].

We evaluated the sensitivity and specificity of an immunochromatography kit, Single-path Emetic Tox Mrk (Merck), which targets a marker protein for the detection of Bacillus cereus that produces emetic toxin. Strains were isolated after outbreaks of food poisoning, and from retail prepared foods and food products. The strains were examined for the presence of the emetic toxin-synthetase gene by PCR. All 58 emetic strains isolated from the food poisoning cases showed a positive reaction in the immunochromatography kit. No emetic strains gave false negative result. Among 47 non-emetic strains, only two strains isolated from the food poisonings and one strain isolated from food products showed a false positive reaction in the test. We concluded that this method has high sensitivity and specificity. The test can be used for detection of emetic toxin-producing B. cereus not only from food poisoning cases, but also in food products.

Genus Enhydrobacter Staley et al. 1987 should be recognized as a member of the family Rhodospirillaceae within the class Alphaproteobacteria.

The genus Enhydrobacter, first reported as a member of the family Vibrionaceae, has been placed in the family Moraxellaceae, but as a genus incertae sedis in Bergey's Manual of Systematic Bacteriology 2nd edition. During our taxonomic investigation of Enhydrobacter-like organisms, we observed that the 16S rRNA sequences of E. aerosaccus-type strain versions NCIMB 12535(T) , ATCC 27094( T) and CCUG 58314(T) were very different from the accessible data (accession no. AJ550856). Phylogenetic analysis of our 16S rRNA sequence data revealed that these organisms were located within the family Rhodospirillaceae. The genera Inquilinus, Oceanibaculum, Skermanella and Nisaea were closely related (sequence similarities were 88.3~87.0%), but Enhydrobacter could be distinguished from these genera by growth characteristics, fatty acid profiles (C(19:0) cyclo ω8c; 38.4% C(18:1) ω7c; 32.2%, and C(16:0) ; 8.9% were major components), in being non-flagellated, and differing in enzymatic activities, including trypsin and ß-glucosidase. From these data, we conclude that the genus Enhydrobacter should be recognized as an independent genus of the family Rhodospirillaceae within the class Alphaproteobacteria.

Intragenomic diversity of the V1 regions of 16S rRNA genes in high-alkaline protease-producing Bacillus clausii spp.

Alkaliphilic Bacillus sp. strain KSM-K16, which produces high-alkaline M-protease, was characterized phenotypically, biochemically and genetically. This strain was identified as Bacillus clausii based on the results of taxonomic studies, including sequencing of the 16S rRNA gene and DNA-DNA hybridization. Seven rRNA operons in the genome were identified by pulsed-field gel electrophoresis. Sequencing of cloned 16S rRNA genes revealed two distinct types of variable region V1. Moreover, some cloned 16S rRNA genes in some of the reference strains of B. clausii had a V1 region of yet another type. The B. clausii strains could clearly be divided into at least two subgroups based on the frequencies of the types of cloned V1 sequence. Bacillus sp. strain KSM-K16 was found to be in a different phylogenetic position from other high-alkaline protease-producing strains of B. clausii.

A novel species of alkaliphilic Bacillus that produces an oxidatively stable alkaline serine protease.

A novel gram-positive, strictly aerobic, motile, sporulating, and facultatively alkaliphilic bacterium designated KSM-KP43 was isolated from a sample of soil. The results of 16S rRNA sequence analysis placed this bacterium in a cluster with Bacillus halmapalus. However, the level of the DNA-DNA hybridization of KSM-KP43 with B. halmapalus was less than 25%. Moreover, the G + C contents of the genomic DNA were 41.6 mol% for KSM-KP43 and 38.6 mol% for B. halmapalus. Because there were also differences in physiological properties and cellular fatty acid composition between the two organisms, we propose KSM-KP43 as a novel species of alkaliphilic Bacillus. This novel strain produces a new class of protease, an oxidatively stable serine protease that is suitable for use in bleach-based detergents. The enzyme contained 640 amino acid residues, including a possible approximately 200-amino-acid prepropeptide in the N-terminal and a unique stretch of approximately 160 amino acids in the C-terminal regions (434-amino-acid mature enzyme with a calculated molecular mass of 45,301 Da). The C-terminal half after the putative catalytic Ser255 and the contiguous C-terminal extension shared local similarity to internal segments of a membrane-associated serine protease of a marine microbial assemblage and the serine protease/ABC transporter precursors of the slime mold Dictyostelium discoideum, and to the C-terminal half of a cold-active alkaline serine protease of a psychrotrophic Shewanella strain.