Diversity and significance of mold species in Norwegian drinking water

In order to determine the occurrence, distribution, and significance of mold species in groundwater- and surface water-derived drinking water in Norway, molds isolated from 273 water samples were identified. Samples of raw water, treated water, and water from private homes and hospital installations were analyzed by incubation of 100-ml membrane-filtered samples on dichloran-18% glycerol agar. The total count (number of CFU per 100 ml) of fungal species and the species diversity within each sample were determined. The identification of mold species was based on morphological and molecular methods. In total, 94 mold species belonging to 30 genera were identified. The mycobiota was dominated by species of Penicillium, Trichoderma, and Aspergillus, with some of them occurring throughout the drinking water system. Several of the same species as isolated from water may have the potential to cause allergic reactions or disease in humans. Other species are common contaminants of food and beverages, and some may cause unwanted changes in the taste or smell of water. The present results indicate that the mycobiota of water should be considered when the microbiological safety and quality of drinking water are assessed. In fact, molds in drinking water should possibly be included in the Norwegian water supply and drinking water regulations.     

Diversity of the microscopic fungi in the littoral sands of the White Sea

For the first time, the study of the mycobiota of littoral sands of the cold-water White Sea was conducted. Twenty-four samples were collected in July–August 2015 in the Onega Bay, the Kandalaksha Bay, and on the Terskii coast. Culture methods were used to detect the number of colony forming units (CFU) and the fungi diversity. The number of CFUs is small; it ranges from 0 to 57 per 1 cm³. A total of 54 morphotypes were detected, of which 22 were identified to the species, seven to the genus level, and 25 were not identified, because they did not form sporulation. Only two species, Cladosorium sphaerospermum and Paradendryphiella salina, were characterized by the relative abundance of more than 10% and the frequency of occurrence of more than 50%. Another five species were found in more than one sample: Penicillium chrysogenum, Cadophora fastigiata, Acremonium fuci, anamorph of Pseudogymnoascus pannorum, and teleomorph of Pseudeurotium hygrophilum. All other morphotypes were found in a small number in single samples. In general, darkcolored forms predominated in the mycobiota both in diversity and in number.     

Assessment of Anthropogenic Influence on Antarctic Mycobiota in Areas of Russian Polar Stations

This article presents the results of microscopic fungi complexes in the areas of five Russian polar stations in East Antarctica and the Subantarctic. A total of 104 microfungal species have been identified. Seventyseven fungal species have been detected in samples of soils and anthropogenic materials from polar stations of East Antarctica (Progress, Mirny, Molodezhnaya, and Druzhnaya 4) using mycological methods while, in the Bellingshausen station (Subantarctic), we have isolated 87 micromycete species. The number of fungi in soils varies from individual propagules in control soils to 94000 per 1 g of soil in contaminated areas. The largest number of species is represented by the genus Penicillium (26 species). Fungal species that form the core of mycobiota in most of the studied habitats have been identified. For soils of East Antarctica, it is formed by species of the genera Aureobasidium, Cadophora, Pseudogymnoascus (Geomyces), Thelebolus, and Phoma. Significant differences are established between the mycobiota of East Antarctica and that of the Subantarctic. At the same time, a general trend towards an increase in fungal species diversity and number in the areas of polar stations compared to the control (clean) sites for all studied areas is recorded. These data indicate that a significant part of micromycetes is introduced into the Antarctic by humans (anthropogenic invasion).     

An original habitat of tempeh molds

Tempeh is a traditional Indonesian food made from soybeans fermented with Rhizopus species. Some researchers believe the original habitat of the tempeh molds may be closely related to fresh leaves of Hibiscus species because these leaves artificially infected with the tempeh molds are used to start tempeh fermentation in cottage-scale factories. To verify this hypothesis, we investigated the occurrence of Rhizopus species in Hibiscus leaves and identified the isolated Rhizopus strains precisely. Rhizopus oryzae, one of the tempeh molds, occurred in sample leaves of some Hibiscus species with considerable frequency. This result implies that tempeh molds that lived in Hibiscus leaves might have fermented soybeans accidentally when used to wrap the cooked soybeans. The original habitat of the tempeh mold could be fresh leaves of Hibiscus species.     

Contamination of common spices in Saudi Arabia markets with potential mycotoxin-producing fungi

Fifteen spices obtained from common markets were examined for their mould profile. A total of 520 fungal isolates, representing 57 species, were recovered and identified from dried and ground spice samples on three different media using standard dilution plate method. The most heavily contaminated spice samples examined were observed in ginger in order of magnitude of 5325–6800 cfu/g. The most predominant fungal genera encountered were Aspergillus, Penicillium, and Rhizopus. Relative occurrence values of taxa disclosed ranged between 80% for Aspergillusflavus, Aspergillusniger and Penicilliumarenicola, and 10% for some species. Samples obtained from sumac encountered very rare colony counts indicating its antifungal prosperities. The present magnitude of contamination and spectra of mycobiota approximate those reported for similar spice samples. Several potentially mycotoxigenic fungi were isolated from the majority of samples. The present study attracts the attention to potential risk for mycotoxins contamination may be caused as a result of using these spices, especially in great quantities. The study strongly recommends reduction in application of heavily contaminated spices like ginger in food processing and using some others like clove and sumac due to their antimicrobial properties.     

Diversity and antibiotic resistance of Acinetobacter spp. in water from the source to the tap

Acinetobacter spp. are ubiquitous bacteria in the environment. Acinetobacter spp. isolated from a municipal drinking water treatment plant and from connected tap water were identified to the species level on the basis of rpoB gene partial sequence analysis. Intraspecies variation was assessed based on the analysis of partial sequences of housekeeping genes (rpoB, gyrB, and recA). Antibiotic resistance was characterized using the disk diffusion method and isolates were classified as wild or non-wild type (non-WT), according to the observed phenotype. The strains of Acinetobacter spp. were related to 11 different validly published species, although three groups of isolates, presenting low rpoB sequence similarities with previously described species, may represent new species. Most of the isolates were related to the species A. johnsonii and A. lwoffii. These two groups, as well as others related to the species A. parvus and A. tjernbergiae, were detected in the water treatment plant and in tap water. Other strains, related to the species A. pittii and A. beijerinckii, were isolated only from tap water. Most of the isolates (80 %) demonstrated wild type (WT) to all of the 12 antibiotics tested. Non-WT for tetracycline, meropenem, and ceftazidime, among others, were observed in water treatment plant or in tap water samples. Although, in general, this study suggests a low prevalence of acquired antibiotic resistance in water Acinetobacter spp., the potential of some species to acquire and disseminate resistance via drinking water is suggested.     

Fate in Model Ecosystems of Microbial Species of Potential Use in Genetic Engineering

Three Oscillatoria strains and one Anabaena species were isolated from three different water supply systems in California that experienced earthy-musty taste and odor problems in their drinking water. Unialgal cultures, free of actinomycetes, were purged using the Grob closed-loop stripping analysis method, and the resulting methylene chloride extracts were analyzed on a gas chromatograph/mass spectrometer. Geosmin was produced by Oscillatoria simplicissima and Anabaena scheremetievi, and 2-methylisoborneol was produced by O. curviceps and O. tenuis. These compounds are the two major causes of earthy-musty tastes and odors in water. In three instances, the major odorant found in culture was previously identified in the water or sediment sample from which the respective organism was isolated. O. curviceps was implicated in a taste and odor episode involving 2-methylisoborneol in a major reservoir. Geosmin and 2-methylisoborneol were easily detected with culture samples of only 4 to 25 ml.     

The Tracing of Mycobacteria in Drinking Water Supply Systems by Culture, Conventional, and Real Time PCRs

Mycobacteria are widely present in diverse aquatic habitats, where they can survive for months or years while some species can even proliferate. The resistance of different mycobacterial species to disinfection methods like chlorination or ozonation could result in their presence in the final tap water of consumers. In this study, the culture method, Mycobacterium tuberculosis complex conventional duplex PCR for detection of non-tuberculous mycobacteria (NTM) and quantitative real-time PCR (qPCR) to detect three subspecies of M. avium species (M. a. avium, M. a. hominissuis, and M. a. paratuberculosis) were used to trace their possible path of transmission from the watershed through the reservoir and drinking water plant to raw drinking water and finally to households. A total of 124 samples from four drinking water supply systems in the Czech Republic, 52 dam sediments, 34 water treatment plant sludge samples, and 38 tap water household sediments, were analyzed. NTM of 11 different species were isolated by culture from 42 (33.9 %) samples; the most prevalent were M. gordonae (16.7 %), M. triplex (14.3 %), M. lentiflavum (9.5 %), M. a. avium (7.1 %), M. montefiorenase (7.1 %), and M. nonchromogenicum (7.1 %). NTM DNA was detected in 92 (76.7 %) samples. By qPCR analysis a statistically significant decrease (P < 0.01) was observed along the route from the reservoir (dam sediments), through water treatment sludge and finally to household sediments. The concentrations ranged from 10⁰ to 10⁴ DNA cells/g. It was confirmed that drinking water supply systems (watershed–reservoir–drinking water treatment plant–household) might be a potential transmission route for mycobacteria.     

The importance of combining air sampling and surface analysis when studying problematic houses for mold biodiversity determination

Houses that underwent water damages are often responsible for heath problems of the occupants. Since there is no universally used protocol for the analysis, we wanted to verify the usefulness of surface sampling versus air sampling for the evaluation of mold diversity in problematic houses and the value of the number of visible mold growth zones to predict air quality. Seventeen houses were sampled for culturable molds in the air and on the surfaces showing contamination. We compared the mold taxa found in the air and on the surfaces and verified the correlation between the number of moldy surfaces and airbone mold concentration. This study demonstrated that, surprisingly, some of the so called wet spore molds (e.g. Stachybotrys) were found more often from air than surface samples whereas, some dry spore molds (e.g. Asp. fumigatus) was more easily isolated from surface samples. There was a good correlation between the number of visible mold growth zones and the concentration of airborne molds. We conclude that air and surface sampling are necessary to evaluate mold diversity in problematic houses and that the number of mold growth zones is a good predictor of airborne mold concentration.     

Effect of Inoculation Techniques and Relative Humidity on the Growth of Molds on the Surfaces of Yellow Layer Cakes

Four inoculation techniques were compared for initiation of growth on cake surfaces: spot, air cabinet, spray (atomizer), and talc addition methods. Molds were isolated from commercial cakes and were identified as Aspergillus sydowii, Aspergillus ochraceus, Penicillium funiculosum, and Eurotium herbariorum. Cake surfaces were inoculated with mold spores and incubated under three equilibrium relative humidity (ERH) levels: 97, 85, and 75%. Random contamination by spores in a ventilated air cabinet was the simplest method of inoculation, but standard deviations in the inoculation rates (20% on a relative scale) were almost twice those observed with the other methods. The spot method was the most reproducible. Cake samples inoculated in the air cabinet had colony counts 10 times lower than those obtained for potato dextrose agar plates at 97% ERH, which was not the case with the spray and talc methods. Growth of molds was much slower in the samples incubated in 75% relative humidity, with all methods. Colony counts were generally similar in systems adjusted at 85 to 97% ERH but were lower for samples incubated at 75% ERH. In comparisons of the shelf life estimates obtained by the various inoculation methods, a correlation coefficient (r²) of 0.70 was obtained between the spot method and the other methods of inoculation, while talc, air cabinet, and spray shelf life data were correlated better (r² ≈ 0.97). The spot method appeared to be the method of choice in consideration of ease of use, precision, and the ability to enable the study of the effects of the environment on mold-free shelf life as well as on the rate of growth of molds on cakes.     

Analysis of the structure of bacteria communities and detection of resistance genes of quinolones from pharmaceutical wastewater

The aim of the present study was to investigate the distribution of bacteria and detect the presence of quinolone resistance gene (qnrA) and integrons (intI1, intI2) in a habitat polluted by pharmaceutical sewage. The bacteria were isolated by nutrient agar and nutrient broth from waste water and sludge collected from the sewage outfall of a pharmaceutical factory. The bacteria were identified by Gram staining and biochemical tests, and the bacterial community diversity was analyzed by Shannon–Wiener diversity index (H), Pielou evenness index (J) and Simpson’s diversity index (D). The occurrence of qnrA and integrons (intI1, intI2) were detected by Real-time PCR assays. The results showed that 90 strains were isolated from water samples and sludge samples including 22 genera and 26 species. Types of bacteria in water samples contained 18 genera and 20 species, while 13 genera and 14 species were detected in sludge samples. Fifty-five Enterobacteriaceae isolates (61.11 %, 55 of 90) were the predominant bacteria in water and sludge samples. Bacterial species richness and evenness in water samples were higher than in sludge samples. The resistance genes of qnrA and integrons (intI1, intI2) with the total DNA and single isolate plasmid DNA were detected. There were a variety of bacterial species and the presence of qnrA and integrons (intI1, intI2) genes in pharmaceutical wastewater habitats, in which Enterobacteriaceae strains were the dominant bacteria. These results suggested that pharmaceutical wastewater had potential risks to public health.     

Durability of rubberwood (Hevea brasiliensis) treated with peppermint oil,eucalyptus oil,and their main components

Anti-fungal activities of two essential oils (peppermint oil and eucalyptus oil) and their main components (menthol and eucalyptol, respectively) against molds (Aspergillus niger, Penicillium chrysogenum, and Penicillium sp.) and a white-rot decay fungus (Trametes versicolor) identified from rubberwood surfaces were investigated. The broth dilution method and the agar diffusion technique were employed to determine the minimal inhibitory concentration (MIC) and the minimal fungicidal concentration (MFC) using the concentration of substances between 100 and 800 μl ml^?1. Inhibitory effects of essential oils and their main components at the MICs against mold growth, fungal decay, and termite attack on rubberwood were further examined by means of the dip treatment method. It was found that MFC values against molds for all treatments examined were about 50–100 μl ml^?1 higher than MIC values. Peppermint oil and menthol exhibited high fungistatic and fungicidal activities, with MICs of 300 μl ml^?1 and 350 μl ml^?1, respectively, against the test molds and the decay fungus. Eucalyptus oil and eucalyptol were also effective against these microbes but at higher concentrations of 600 μl ml^?1 and 500 μl ml^?1, respectively. Only peppermint oil at the MIC was capable of providing a complete protection from mold growth on rubberwood for up to 12 weeks at storage conditions of 25 °C and 100% RH. Both peppermint oil and eucalyptus oil at the MICs showed moderate resistance to fungal decay and high resistance to termite attack.     

Taxonomic re-evaluation of black koji molds

Black koji molds including its albino mutant, the white koji mold, have been widely used for making the distilled spirit shochu in Northeast Asia because they produce citric acid which prevents undesirable contamination from bacteria. Since Inui reported Aspergillus luchuensis from black koji in Okinawa in 1901, many fungal names associated with black koji molds were reported. However, some species are similar and differentiation between species is difficult. Fungal taxonomists tried to arrange a taxonomic system for black koji molds, but the results were not clear. Recently, multi-locus sequence typing has been successfully used to taxonomy of black Aspergillus. According to β-tubulin and calmodulin gene sequences, black koji molds can be subdivided in three species, A. luchuensis, Aspergillus niger, and Aspergillus tubingensis. Aspergillus awamori, Aspergillus kawachii, Aspergillus inuii, Aspergillus nakazawai, and Aspergillus coreanus are synonyms of A. luchuensis, Aspergillus batatae, Aspergillus aureus (or Aspergillus foetidus), Aspergillus miyakoensis, and Aspergillus usamii (including A. usamii mut. shirousamii) are synonyms of A. niger and Aspergillus saitoi and A. saitoi var. kagoshimaensis are synonyms of A. tubingensis. A. luchuensis mut. kawachii was suggested particular names for A. kawachii because of their industrial importance. The history and modern taxonomy of black koji molds is further discussed.     

Pyrosequence Analysis of the hsp65 Genes of Nontuberculous Mycobacterium Communities in Unchlorinated Drinking Water in the Netherlands

Studies have shown that certain opportunistic pathogenic species of nontuberculous mycobacteria (NTM) can be present in distributed drinking water. However, detailed information about NTM population composition in drinking water is lacking. Therefore, NTM communities in unchlorinated drinking water from the distribution system of five treatment plants in the Netherlands were characterized using 454 pyrosequencing of the hsp65 gene. Results showed high diversities in unchlorinated drinking water, with up to 28 different NTM operational taxonomic units (OTUs) in a single sample. Each drinking water sample had a unique NTM community, and most (81.1%) OTUs were observed only once. One OTU was observed in 14 of 16 drinking water samples, indicating that this NTM species is well adapted to unchlorinated drinking water conditions. A clear influence of season, source type (groundwater, surface water), easily assimilable organic carbon (AOC) concentration, biofilm formation rate, and active biomass in treated water on the establishment of an NTM community in drinking water was not observed. Apparently, local conditions are more important for the development of a specific NTM community in the drinking water distribution system. A low (4.2%) number of hsp65 gene sequences showed more than 97% similarity to sequences of the opportunistic pathogens M. avium, M. genavense, and M. gordonae. However, most (95.8%) NTM hsp65 gene sequences were related to not-yet-described NTM species that have not been linked to disease, indicating that most NTM species in unchlorinated drinking water from distribution systems in the Netherlands have a low public health significance.     

Molds in Brined Cucumbers: Cause of Softening During Air-Purging of Fermentations

Softening of cucumbers in fermentations purged at high air-flow rates was caused by molds growing in the brined cucumbers, not in the brine. This conclusion is based on the following results: (i) no microorganisms were isolated in significant numbers from brines that caused softening of pasteurized brined cucumbers, (ii) no pectinolytic enzyme activities were produced in cucumber brines in the absence of cucumbers, (iii) the pickles in some air-purged fermentations became very soft without the appearance of any pectinolytic enzyme activity in the brine, (iv) mold hyphae were consistently observed in tissues of soft pickles, (v) molds consistently developed in cultures of slices of surface sterilized cucumbers taken from fermentations in which soft pickles were subsequently found, and (vi) molds belonging to the genera Alternaria, Fusarium, and Mucor isolated from slices all softened pasteurized brined cucumbers.     

Diversity of the endophytic fungi associated with the ancient and narrowly endemic neotropical plant Vellozia gigantea from the endangered Brazilian rupestrian grasslands

This study focused on the taxonomy and diversity of the endophytic fungi associated with Vellozia gigantea, an endemic, ancient, and endangered plant species that occurs only in the rupestrian grasslands of Brazil. A total of 285 fungal isolates were recovered from leaves and roots of the V. gigantea, which were identified in 27 genera and 87 different taxa using molecular taxonomy methods. Xylaria berteri, Diaporthe sp. 1, Nigrospora oryzae, Muscodor sp. 1, Colletotrichum aeschynomene, and Trichoderma viride occurred in the highest frequency in both the leaf and root. Diaporthe was the most abundant genus, with 70 endophytic isolates recovered from the leaves and roots. Among all the taxa identified, 62 occurred as singlets, including those of the genera Clonostachys, Coccomyces, Crucellisporiopsis, Daldinia, Myxotrichum, Pallidocercospora, Pezicula, Peyronellaea, and Pseudocercospora. The diversity indices displayed high values, showing that V. gigantea shelters a diverse and rich mycobiota. Our results indicate that V. gigantea shelters in its tissues a highly diverse and cryptic mycobiota, including several rare species previously unreported as endophytes, but that are reported to have different ecological functions, which might be an important biological component contributing to the fitness of the plants living in the rupestrian grassland.     

Characterization of fungi associated with the nasal hairs of Molossid bats

Fungal pathogens have become a serious threat to wildlife, threatening populations of even once common, abundant species. We describe the mycobiota associated with the nasal hairs of three Molossid bat species, Cynomops planirostris, Molossus molossus, and Molossus rufus, in southwest Brazil. Bats were captured in the Cerrado and Pantanal biomes. We cultured 22 fungal isolates from twelve individual bats. Sixteen sequences of the ITS region were obtained, yielding 11 unique sequence types from the genera Aspergillus, Cladosporium, Paecilomyces, and Penicillium. No obvious detrimental effects on the bats from the fungi were observed, although some species or genera that we identified are known pathogens in other species. This is the first report of such fungi associated with the nasal hairs of Molossid bats. Our results indicate the need for further research on the biodiversity, ecological role, and potential effects of this mycobiota on Molossid bats.     

Nontuberculous mycobacteria: an emerging risk in engineered environmental habitats

Nontuberculous mycobacteria (NTM) are microorganisms commonly living in the environment. Nevertheless, most of them are opportunistic pathogens. To verify concentrations of NTM in some man-made habitats, analyses were performed on water and surface samples, and the data were correlated to the global microbiological quality of water. Most of the drinking water samples (98 %) complied with the microbial requirements established by the European Directive 98/83/EC on drinking water when Escherichia coli was considered. Low counts of heterotrophs were also obtained. NTM were isolated from 72 % of the analyzed samples. Tap water from private buildings, schools and hospitals provided positive results for NTM, with comparable densities ranging from 1 to 6?×10² CFU/L. NTM were also found in swimming pool water samples, with concentrations ranging from 29 to 3?×10⁴ CFU/L, as well as in 70 % of the surface sample. The most frequently isolated species were Mycobacterium mucogenicum, M. intracellulare and M. terrae. These yields confirm that no correlation exists between the monitoring controls carried out by law and the occurrence of these bacteria that may represent a potential risk, especially for immunocompromised people and vulnerable groups.     

Pilot study of mold populations inside and outside of Puerto Rican residences

Puerto Rico has the highest asthma prevalence in the US. In the states, mold exposures have been linked to the development and exacerbation of asthma. For a pilot study of mold populations in Puerto Rico, dust and air samples were collected in January 2013 inside and outside of residences (n = 10) in the north eastern quadrant of Puerto Rico. Inside and outside (protected from rain) settled dust was collected by wiping above floor or ground-level surfaces with a Swiffer? duster cloth. Air samples were collected in five of the residences inside and five matching outside onto Malt Extract Agar and also onto G25N Agar. The dust (5 mg) was analyzed using mold-specific quantitative PCR and mold contamination estimated using the Environmental Relative Moldiness Index (ERMI) metric. Air sampled (150 l) plates were incubated and resulting mold colonies enumerated. The ERMI values were significantly (p = 0.03) greater inside the homes than outside, 20.37 versus 12.55, respectively. Aspergillus penicillioides, A. sydowii, Chaetomium globosum, the Eurotium group and Penicillium crustosum and A. ustus were found in significantly greater numbers inside than outside Puerto Rican residences based on the Wilcoxon rank sum test. However, after correction for multiple comparisons using the Holms–Bonferroni test, only the Eurotium group remained significantly greater. Air samples suggested higher culturable concentrations of molds outside but the differences were not significantly different. Although this is a small study, it suggests that some people in Puerto Rico are exposed to significantly higher mold concentrations indoors than outdoors.     

The Black Yeast Exophiala dermatitidis and Other Selected Opportunistic Human Fungal Pathogens Spread from Dishwashers to Kitchens

We investigated the diversity and distribution of fungi in nine different sites inside 30 residential dishwashers. In total, 503 fungal strains were isolated, which belong to 10 genera and 84 species. Irrespective of the sampled site, 83% of the dishwashers were positive for fungi. The most frequent opportunistic pathogenic species were Exophiala dermatitidis, Candida parapsilosis sensu stricto, Exophiala phaeomuriformis, Fusarium dimerum, and the Saprochaete/Magnusiomyces clade. The black yeast E. dermatitidis was detected in 47% of the dishwashers, primarily at the dishwasher rubber seals, at up to 10⁶ CFU/cm²; the other fungi detected were in the range of 10² to 10⁵ CFU/cm². The other most heavily contaminated dishwasher sites were side nozzles, doors and drains. Only F. dimerum was isolated from washed dishes, while dishwasher waste water contained E. dermatitidis, Exophiala oligosperma and Sarocladium killiense. Plumbing systems supplying water to household appliances represent the most probable route for contamination of dishwashers, as the fungi that represented the core dishwasher mycobiota were also detected in the tap water. Hot aerosols from dishwashers contained the human opportunistic yeast C. parapsilosis, Rhodotorula mucilaginosa and E. dermatitidis (as well as common air-borne genera such as Aspergillus, Penicillium, Trichoderma and Cladosporium). Comparison of fungal contamination of kitchens without and with dishwashers revealed that virtually all were contaminated with fungi. In both cases, the most contaminated sites were the kitchen drain and the dish drying rack. The most important difference was higher prevalence of black yeasts (E. dermatitidis in particular) in kitchens with dishwashers. In kitchens without dishwashers, C. parapsilosis strongly prevailed with negligible occurrence of E. dermatitidis. F. dimerum was isolated only from kitchens with dishwashers, while Saprochaete/Magnusiomyces isolates were only found within dishwashers. We conclude that dishwashers represent a reservoir of enriched opportunistic pathogenic species that can spread from the dishwasher into the indoor biome.