An experimental verification of fungal overgrowth in temporary houses at the site of the Great East Japan Earthquake.

Fungal contamination in the indoor air of prefabricated temporary houses at the site of the Great East Japan Earthquake revealed extremely high levels compared to those found in conventional residences. We experimentally investigated fungal growth levels on different interior materials to support fungal overgrowth in prefabricated temporary houses. Three species each of allergenic fungi and invasive fungi observed in temporary housing were selected for inoculation tests with various interior materials. The experiments with fungal inoculation were conducted in conformance with standards for industrial products described in the Japanese" JIS Z 2911:2018 Methods of test for fungus resistance" with small modifications. After incubation, visual and stereomicroscopic assessments were performed to determine fungal growth levels. The viability of the fungi varied according to the interior material type. Our findings demonstrate the importance of antifungal measures in indoor environments and the need for additional research on the growth levels of fungal species on various interior materials.

Indoor Fungal Contamination in Temporary Housing after the East Japan Great Earthquake Disaster.

To understand fungal contamination in the indoor environment of the disaster region, a field survey was performed to measure the number of fungal counts and identify isolates in the indoor air of prefabricated temporary housing, privately independent-housing, and rented apartments flooded by the East Japan Great Earthquake disaster tsunami. As a result, the period with the highest detected fungal count was from the rainy season to summer in independent-housing and rented apartments. Moreover, in the temporary housing, the fungal number increased further in winter as indicated by the maximum fungal-number throughout the measurement period. The detection frequency of Aspergillus species was relatively higher in the indoor air of temporary housing than in typical housing in the non-disaster area. Since Aspergillus is known as an allergenic genus, it requires careful attention to the health risk for residents. The extremely high level of fungal condensation in indoor air possibly occurred due to high relative humidity and loss of heat insulation in the building attics. It is suggested that this problem commonly happened in the cold region including the entire disaster region of the East Japan Great Earthquake.

A New Protocol for the Detection of Sterigmatocystin-producing Aspergillus Section Versicolores Using a High Discrimination Polymerase.

Aspergillus section Versicolores species, except Aspergillus sydowii, produce a carcinogenic mycotoxin sterigmatocystin (STC). Since these fungi are found in varied environmental milieu including indoor dust and food products, our aim was to develop a sensitive and convenient assay to detect STC producing fungal strains. We made use of a high discrimination DNA polymerase (HiDi DNA polymerase), for single nucleotide polymorphism (SNP)-based PCR amplification. Using specific primer pairs based on the SNPs between A. sydowii and other strains of Aspergillus section Versicolores, we succeeded in amplifying the genomic DNA all target strains except A. sydowii. These results confirm that the SNP-based PCR amplification technique, using a high discrimination DNA polymerase, was a reliable and robust screening method for target fungal strains.

Effects of UV Irradiation on Penicillium Strains Isolated from a Bread Plant and the Application to Bakery Products.

We tested treatement with UV irradiation for controlling the growth of bread mold. First, we analyzed the sterilizing effect of a dose of approximately 25 mJ/cm2 radiation on nine Penicillium and two Talaromyces strains that were isolated from a bread-manufacturing plant. The P. chermesinum and P. paneum strains were sterilized completely at that dose, while it was only partially effective against P. corylophilum. P. chrysogenum and P. decumbens were sterilized at a dose of approximately 120 mJ/cm2, while T. amestolkiae was sterilized at approximately 150 mJ/cm2. Sterilization of T. cecidicola and P. hispanicum required more than 200 mJ/cm2 of radiation. These results suggest that UV resistance varies depending on the species and the strains. We also carried out UV irradiation of bread at 70 mJ/cm2: a dose at which the taste of bread is not affected; we observed that mold growth was delayed visibly compared to the non-irradiated bread. These results suggest that UV irradiation at 70 mJ/cm2 is effective at delaying mold growth, though it does not cause complete sterilization. This method should prove useful for extending the shelf-life of bread.

New Insight on Fumigation Action of Essential Oil, Commercial Fungicide and Low Oxygen Microenvironment on Museum Mold, Alternaria alternata.

Fumigation has been the most convenient method in the field of pest control in museums. In this study, as fumigants, ethanol 70%, deltamethrin (commercial pesticide (CP) ) , essential oil (EO) from Pinus regida, and low oxygen microenvironment (0.1%, (LOM) ) were tested individually and jointly against museum fungal strain Alternaria alternata. Three concentrations of each CP and EO were chosen for evaluating the individual effect. In the joint action fumigation process, three lower concentrations of CP and EO were tested in LOM. The rate of mycelial growth inhibition at each fumigation process was determined by two steps: 1) directly after the fumigation process and 2) after 7 d of the inoculation of the fumigated spores in new medium and incubating it in normal condition. The results demonstrated that applying of each chemical (CP or EO) in LOM enhanced its fungicidal activity and that effect of EO improved from fungistatic to fungicidal by jointing with LOM.

Fungicidal Effects of Ultraviolet Light (254 nm) Irradiation on Contaminated Museum Packing and Storing Materials.

In storage of modern museums, collections are packed and stored with acid-free paper-based materials for keeping safe and stable conditions. Direct contact of fungal contaminated packing and storing materials with the collections is concerned about expanding of infection in storage facilities. In this study, fungicidal effects of UV light irradiation on the materials such as archival board and Japanese tissue paper contaminated with Penicilliun commune and Chaetomium globosum were tested. The analyzed materials were divided into two groups; Group 1 was examined with 20 µl of spore suspensions of fungi (106 cfu/ml) ; and Group 2 was tested on Czapek- Dox agar medium modified without sugar and inoculated with 100 µl of the spore suspensions of fungi (106 cfu/ml) . Six doses of UV irradiation were examined on Group 1 and five doses on Group 2 in addition to control. The assessment was done by using 1) adenosine triphosphate (ATP) bioluminescence assay and double staining to determine the cell viability; 2) observation under light microscope to evaluate morphophysiological change of tested fungi (spores and hyphae) . Because of the thinness and high transparency of tissue paper, UV irradiations were highly efficient to fungicide its fungal contamination compared with archival board. In spite of the high resistance of C. globosum spores, the rate of growth was slow, and with a little amount of perithecia or fruiting bodies and a high amount of ycelium (which damaged rapidly through UV irradiation) . This may be due to a low relative humidity of the incubation environment. Minimum dosage of UV irradiation with fungicidal effectiveness against all fungal contamination was estimated as 118 J/cm2.

Fatal pneumonia caused by Penicillium digitatum: a case report.

BACKGROUND: Penicillium species are among the most common fungi present in the environment and are usually considered non-pathogenic to humans. However, in immunocompromised hosts they can be virulent pathogens and can cause death. Penicillium digitatum is a plant pathogen that commonly causes a postharvest fungal disease of citrus called green mould; it very rarely causes systemic mycosis in humans. Here, we report a case of fatal pneumonia due to P. digitatum infection, as confirmed by repeated examination of cultured sputum. CASE PRESENTATION: A cavity was found in the left upper lung on routine chest X-ray in a 78-year-old undernourished male who had been diagnosed at age 66 with bronchial asthma and pulmonary emphysema. No increased sputum production was present. The presence of antigen-specific precipitating antibodies to Aspergillus flavus and P. digitatum was confirmed in the patient's serum and also later pleural fluid by using Ouchterlony double immunodiffusion testing with A. flavus and P. digitatum antigens. The patient was treated over a period of months with itraconazole, micafungin, voriconazole, amphotericin B, and antibacterials. However, the cavity enlarged, the pleural effusion increased, and the patient began producing purulent sputum. He died from progressive renal failure. From sputum culture only one fungus was isolated repeatedly on potato-dextrose agar in large quantities. This fungus was confirmed to be P. digitatum by molecular identification. Partial sequences of the beta-tubulin gene were determined by using the primers Bt2a and Bt2b for PCR amplification and sequencing and underwent a BLAST search at the National Centre for Biotechnology Information, these results confirmed that the isolated fungus was P. digitatum. CONCLUSION: To our knowledge, this is the first report of pulmonary infection with P. digitatum. Our patient had pulmonary emphysema and was elderly, and undernourished. These factors might have facilitated the infection. In his case, antimycotics were ineffective in treating the lung involvement. Although human infection with P. digitatum is considered rare, it appears that this organism can be very virulent and resistant to antimycotics.

Rapid detection of Shiga toxin-producing Escherichia coli in ground beef by an immunochromatography kit in combination with short-term enrichment and treatment for Shiga toxin release.

To establish rapid methods to detect Shiga toxin (Stx)-producing Escherichia coli (STEC) in ground beef samples by using an immunochromatography kit, results of 8-h enrichment in various types of broth with shaking were compared. In pure culture, Stx was detected in the culture of trypticase soy broth (TSB) at 42°C and modified EC broth (mEC) at 36°C from all or most serogroups of O26, O111, O128, O157 and OUT. Ground beef samples inoculated with each serogroup were enriched in TSB at 42°C, mEC at 36°C and mEC with novobiocin (NmEC) at 42°C. Although all conditions led to the successful recovery of each serogroup by the plating method, enrichment in NmEC was relatively superior to the other conditions in the detection of Stx by an immunochromatography kit. These results indicated that the growth of STEC and the release of Stx from cells were different in pure cultures and in culture with ground beef. In addition, polymyxin B treatment for 10 min at 37°C and homogenizing with glass beads enhanced the detection of Stx. From the results, it was suggested that an immunochromatography kit in a combination with enrichment in NmEC at 42°C for 8 h, and treatment with polymyxin B or homogenizing would be a rapid method to detect STEC contamination in ground beef.

Quantitative analysis of mycoflora on commercial domestic fruits in Japan.

A comprehensive and quantitative analysis of the mycoflora on the surface of commercial fruit was performed. Nine kinds of fruits grown in Japan were tested. Overall fungal counts on the fruits ranged from 3.1 to 6.5 log CFU/g. The mean percentages of the total yeast counts were higher than those of molds in samples of apples, Japanese pears, and strawberries, ranging from 58.5 to 67.0%, and were lower than those of molds in samples of the other six fruits, ranging from 9.8 to 48.3%. Cladosporium was the most frequent fungus and was found in samples of all nine types of fruits, followed by Penicillium found in eight types of fruits. The fungi with the highest total counts in samples of the various fruits were Acremonium in cantaloupe melons (47.6% of the total fungal count), Aspergillus in grapes (32.2%), Aureobasidium in apples (21.3%), blueberries (63.6%), and peaches (33.6%), Cladosporium in strawberries (38.4%), Cryptococcus in Japanese pears (37.6%), Penicillium in mandarins (22.3%), and Sporobolomyces in lemons (26.9%). These results demonstrated that the mycoflora on the surfaces of these fruits mainly consists of common pre- and postharvest inhabitants of the plants or in the environment; fungi that produce mycotoxins or cause market diseases were not prominent in the mycoflora of healthy fruits. These findings suggest fruits should be handled carefully with consideration given to fungal contaminants, including nonpathogenic fungi, to control the quality of fruits and processed fruit products.

Microbe-I: fungal biota analyses of the Japanese experimental module KIBO of the International Space Station before launch and after being in orbit for about 460 days.

In addition to the crew, microbes also find their way aboard the International Space Station (ISS). Therefore, microbial monitoring is necessary for the health and safety of the crew and for general maintenance of the facilities of this station. Samples were collected from three sites in the Japanese experimental module KIBO on the ISS (air diffuser, handrail, and surfaces) for analysis of fungal biota approximately 1 year after this module had docked with the ISS. Samples taken from KIBO before launch and from our laboratory were used as controls. In the case of KIBO, both microbe detection sheet (MDS) and swab culture tests of orbital samples were negative. The MDS were also examined by field emission-scanning electron microscopy; no microbial structures were detected. However, fungal DNAs were detected by real-time PCR and analyzed by the clone library method; Alternaria sp. and Malassezia spp. were the dominant species before launch and in space, respectively. The dominant species found in specimens from the air conditioner diffuser, lab bench, door push panel, and facility surfaces on our laboratory (ground controls) were Inonotus sp., Cladosporium sp., Malassezia spp., and Pezicula sp., respectively. The fungi in the KIBO were probably derived from contamination due to humans, while those in our laboratory came from the environment (e.g., the soil). In conclusion, the cleanliness in KIBO was equivalent to that in a clean room environment on the ground.

Mortierella wolfii keratomycosis in a horse.

PURPOSE: To describe a case of superficial keratomycosis caused by Mortierella wolfii (M. wolfii) in a horse. METHODS: A thoroughbred filly was presented with painful right eye of 2 days' duration. A superficial corneal ulcer was observed ventrally together with multifocal punctuate opacities axially. Samples were collected by swabbing and scraping the ulcerated lesion and submitted for microbiologic and cytologic examination. RESULTS: Microscopic evaluation of debrided corneal tissue revealed the presence of nonseptate fungal hyphae, and culture of a corneal swab yielded fungal growth. Medical treatment with topical antifungal, antibiotic and autogenous serum and systemic anti-inflammatory resolved the problem within 2 weeks. CONCLUSIONS: Cytologic evaluation of a corneal scraping was useful to make a clinical diagnosis of keratomycosis. Based on the mycological characteristics, the fungus isolated from the corneal lesion was identified as M. wolfii. To the authors' knowledge, this is the first case report of equine keratomycosis associated with this fungus, although the organism is known to infect various organs of cattle.

Insights into a nonhomologous integration pathway in the dermatophyte Trichophyton mentagrophytes: efficient targeted gene disruption by use of mutants lacking ligase IV.

Targeted gene disruption experiments in Trichophyton mentagrophytes are impeded by the dominant of repair of DNA double strand breaks through a nonhomologous end joining pathway (NHEJ). Inactivation of human DNA ligase IV homologs, which is involved in the final step of the NHEJ pathway, has been shown to enhance homologous recombination (HR) frequency in filamentous fungi. To improve the frequency of HR in T. mentagrophytes, the lig4 homolog (TmLIG4) was disrupted. T. mentagrophytes lacking TmLIG4 showed no discernable phenotypic differences when compared to wild-type controls. Both mutant and parent strains had almost identical growth ability, sporulation rate and sensitivity to DNA damaging agents. When four different loci were disrupted in the TMLIG4-deficient mutant, HR frequencies reached as high as 93% depending on the locus, whereas they ranged from 0%-40% in the wild-type. These results suggest that studies in strains lacking TmLIG4 would help to improve our understanding of dermatophytosis by facilitating the genetic manipulation of dermatophytes.

Comparison of detection methods for Escherichia coli O157 in beef livers and carcasses.

Beef organ meat, such as liver, and beef are major food sources contaminated with Escherichia coli O157. This study investigated the detection method of E. coli O157 in beef liver and carcass. In an experiment with beef liver inoculated with E. coli O157, the direct plating method, plating after the immunomagnetic separation (IMS) method, and Shiga toxin (Stx)-producing E. coli detection and E. coli O157 detection loop-mediated isothermal amplification (LAMP) assays were compared for the detection of Stx-producing E. coli O157. Fifty percent and 45% of samples were positive by Stx-producing E. coli detection LAMP assay and E. coli O157 detection LAMP assay, respectively. Thirty-five percent and 10% of samples were positive by the IMS method and direct plating method, respectively. In an examination of beef swab samples, contamination frequencies with E. coli O157 were analyzed by LAMP assays and the IMS method. E. coli O157 was detected in 12 of 230 samples (5.2%). There was no sample positive for E. coli O157 isolation but negative for LAMP assays for Stx gene and O157 antigen gene. Four samples (1.7%) were positive by both LAMP assays but negative by the IMS method. The result that there was no sample positive for the O157 antigen gene, but not the Stx gene, indicated that the IMS method failed to detect E. coli O157. Twenty-nine samples (12.6%) were positive for the Stx gene but not the O157 antigen gene. The results indicated that screening of Stx gene and O157 antigen gene by LAMP assays is effective in saving time and effort to isolate E. coli O157 by the IMS method because the LAMP assay is more sensitive. This suggested that samples positive for Stx gene and O157 antigen gene should be examined by the IMS method to isolate E. coli O157.

Enumeration of fungi in fruits by the most probable number method.

In this study, enumeration methods for fungi in foods were evaluated using fruits that are often contaminated by fungi in the field and rot because of fungal contaminants. As the test methods, we used the standard most probable number (MPN) method with liquid medium in test tubes, which is traditionally used as the enumeration method for bacteria, and the plate-MPN method with agar plate media, in addition to the surface plating method as the traditional enumeration method for fungi. We tested 27 samples of 9 commercial domestic fruits using their surface skin. The results indicated that the standard MPN method showed slow recovery of fungi in test tubes and lower counts than the surface plating method and the plate-MPN method in almost all samples. The fungal count on the 4th d of incubation was approximately the same as on the 10th d by the surface plating method or the plate-MPN method, indicating no significant differences between the fungal counts by these 2 methods. This result indicated that the plate-MPN method had a number agreement with the traditional enumeration method. Moreover, the plate-MPN method has a little laborious without counting colonies, because fungal counts are estimated based on the number of plates with growing colonies. These advantages demonstrated that the plate-MPN method is a comparatively superior and rapid method for enumeration of fungi.

Nourseothricin acetyltransferase: a new dominant selectable marker for the dermatophyte Trichophyton mentagrophytes.

Dermatophytes are filamentous fungi that colonize the keratinized layer of human and animal skin. The availability of several selectable markers for dermatophyte gene manipulation would provide important tools to understand the genetic properties of this fungal group. In this study, we report the nourseothricin resistance gene nat1 that confers resistance to the aminoglycoside antibiotic nourseothricin as a dominant marker in Trichophyton mentagrophytes. The NAT cassette was introduced into T. mentagrophytes by the Agrobacterium tumefaciens-mediated transformation (ATMA) method. Transformation occurred at a frequency of 78 transformants per 1 x 10(7) cells. Molecular analysis showed integration of the NAT cassette into the genomic DNA of T. mentagrophytes. This study presents the nourseothricin resistance gene nat1 as a useful selectable marker for selection of T. mentagrophytes.

A suggested pathogenic role for Trichosporon montevideense in a case of Onychomycosis in a Japanese monkey.

Onychomycosis is a fungal infection of fingernails or toenails caused by several species of fungi and yeasts. A Japanese monkey, Macaca fuscata, displayed severe onychomycosis in his 4 limbs. Diagnosis and etiological agent identification were performed by conventional and DNA-mediated methods. The accumulated findings of this case revealed Trichosporon montevideense, which has long been considered to be a nonpathogenic yeast. Here, we present the first report of an involvement of T. montevideense in an onychomycosis case.

[Survival of Salmonella Weltevreden and S. Senftenberg in black tiger shrimp under frozen storage].

Survival of Salmonella in black tiger shrimps during frozen storage was investigated following our previous study on Salmonella contamination in frozen shrimps imported into Japan. Salmonella (S.) Weltevreden and S. Senftenberg were inoculated onto the surface and inside of black tiger shrimps without the shell. After storage at -10 degrees C, -20 degrees C and -30 degrees C for 12 weeks, the Salmonella population decreased in all cases; the decrease was smallest at the lowest temperature. Viability of Salmonella inoculated onto the surface of the shrimp was greater than that inside the shrimp. In addition, viability of S. Senftenberg was greater than that of S. Weltevreden. These results suggest that hygienic handling during thawing of shrimps is important from the viewpoint of Salmonella contamination.

Microbial quality of liquid egg and salmonella infection status in Japan.

Aerobic bacteria counts and contamination with Salmonella were investigated in a total of 1,327 samples of commercial liquid egg in 1992-2002. Salmonella was isolated from 8.1% of the samples, and Salmonella contamination was found in 1.7% of even the pasteurized liquid egg samples. The major Salmonella serotype was Enteritidis from more than 50% of the contaminated liquid egg samples. In addition, the aerobic bacteria counts in Salmonella-positive liquid eggs were significantly higher than those of Salmonella-negative samples. However, Salmonella was detected in liquid egg in which the aerobic bacteria counts were in the range of 10(2) to 10(6) cfu/g. Furthermore, foodborne outbreaks of Salmonella infections associated with liquid egg were analyzed. Liquid eggs should be carefully treated to avoid the possibility of Salmonella contamination. Adequate supply of pasteurized liquid eggs and controls to prevent re-contamination are needed.

Direct colony PCR of several medically important fungi using Ampdirect plus.

Direct colony polymerase chain reaction (DCPCR) is a useful molecular biological technique for application in the field of mycology. In this study, all of the 63 fungal strains examined, including those of the genera Candida and Aspergillus, were amenable to DNA amplification using an Ampdirect(R) Plus kit, which allows direct PCR amplification with no requirement for DNA extraction, following 1 h of rapid fungal lysis. Moreover, we compared DCPCR of 35 strains, representing 20 species, using Ampdirect PCR and standard PCR with no lysis buffer. Thirty-four of these strains (97.14%) yielded positive results on Ampdirect PCR, while only 11 (including Aspergillus fumigatus TIMM1776) of the 35 strains (31.43%) showed PCR products when standard PCR reagents were used. Ampdirect DCPCR was also applicable to DNA amplification for spore and hyphal cells. This approach reduces DNA template preparation time before PCR from fungal colonies, and also reduces the cost of PCR.