Analysis of Black Fungal Biofilms Occurring at Domestic Water Taps (II): Potential Routes of Entry

Formation of tenacious and massive black biofilms was occasionally observed at the water–air interphase of water taps and in associated habitats at several locations in Germany. Exophiala lecanii-corni was proven to be the dominant component of these biofilms. Water utility companies were interested to understand by which route fungi building these black biofilms enter their habitat at affected sites in domestic sanitary. A wide variety of fungi is known to be common in wet indoor environments, as well as in the drinking water resources. Two possible routes of entry are therefore considered as follows: (a) distribution by the drinking water system or (b) a retrograde route of colonisation. Previous compositional analysis revealed that the black constituents of biofilms primarily belong to the herpotrichiellaceous black yeast and relatives. Therefore, a systematic search for black fungi in the drinking water system was performed using Sabouraud’s glucose agar medium with chloramphenicol and erythritol–chloramphenicol agar as isolation media. Cadophora malorum was the dominant fungus in the investigated drinking water systems, and samples taken from the house connections (n = 50; 74 %, <200 cfu/L), followed by a so far undescribed Alternaria sp. (28 %; <10 cfu/L) and E. castellanii (26 %; <10 cfu/L). Of note, C. malorum was not present in any previously analysed biofilm. Since E. lecanii-corni was not found in any water sample from the distribution system tested, but represented the most abundant species in dark biofilms previously analysed, a retrograde route of contamination in case of E. lecanii-corni can be assumed.     

Effect of Phosphorus on Survival of Escherichia coli in Drinking Water Biofilms

The effect of phosphorus addition on survival of Escherichia coli in an experimental drinking water distribution system was investigated. Higher phosphorus concentrations prolonged the survival of culturable E. coli in water and biofilms. Although phosphorus addition did not affect viable but not culturable (VBNC) E. coli in biofilms, these structures could act as a reservoir of VBNC forms of E. coli in drinking water distribution systems.     

Temporal Variations in the Abundance and Composition of Biofilm Communities Colonizing Drinking Water Distribution Pipes

Pipes that transport drinking water through municipal drinking water distribution systems (DWDS) are challenging habitats for microorganisms. Distribution networks are dark, oligotrophic and contain disinfectants; yet microbes frequently form biofilms attached to interior surfaces of DWDS pipes. Relatively little is known about the species composition and ecology of these biofilms due to challenges associated with sample acquisition from actual DWDS. We report the analysis of biofilms from five pipe samples collected from the same region of a DWDS in Florida, USA, over an 18 month period between February 2011 and August 2012. The bacterial abundance and composition of biofilm communities within the pipes were analyzed by heterotrophic plate counts and tag pyrosequencing of 16S rRNA genes, respectively. Bacterial numbers varied significantly based on sampling date and were positively correlated with water temperature and the concentration of nitrate. However, there was no significant relationship between the concentration of disinfectant in the drinking water (monochloramine) and the abundance of bacteria within the biofilms. Pyrosequencing analysis identified a total of 677 operational taxonomic units (OTUs) (3% distance) within the biofilms but indicated that community diversity was low and varied between sampling dates. Biofilms were dominated by a few taxa, specifically Methylomonas, Acinetobacter, Mycobacterium, and Xanthomonadaceae, and the dominant taxa within the biofilms varied dramatically between sampling times. The drinking water characteristics most strongly correlated with bacterial community composition were concentrations of nitrate, ammonium, total chlorine and monochloramine, as well as alkalinity and hardness. Biofilms from the sampling date with the highest nitrate concentration were the most abundant and diverse and were dominated by Acinetobacter.     

Mycobacterium avium Complex in Day Care Hot Water Systems,and Persistence of Live Cells and DNA in Hot Water Pipes

The Mycobacterium avium complex (MAC) is a group of opportunistic human pathogens that may thrive in engineered water systems. MAC has been shown to occur in drinking water supplies based on surface water, but less is known about the occurrence and persistence of live cells and DNA in public hot water systems based on groundwater. In this study, we examined the occurrence of MAC in hot water systems of public day care centers and determined the persistence of live and dead M. avium cells and naked DNA in model systems with the modern plumbing material cross-linked polyethylene (PEX). The occurrence of MAC and co-occurrence of Legionella spp. and Legionella pneumophila were determined using cultivation and qPCR. Co-occurrences of MAC and Legionella were detected in water and/or biofilms in all hot water systems at temperatures between 40 and 54 °C. Moderate correlations were observed between abundance of culturable MAC and that of MAC genome copies, and between MAC and total eubacterial genome copies. No quantitative relationship was observed between occurrence of Legionella and that of MAC. Persistence in hot water of live and dead M. avium cells and naked DNA was studied using PEX laboratory model systems at 44 °C. Naked DNA and DNA in dead M. avium cells persisted for weeks. Live M. avium increased tenfold in water and biofilms on PEX. The results suggest that water and biofilms in groundwater-based hot water systems can constitute reservoirs of MAC, and that amplifiable naked DNA is relatively short-lived, whereas PEX plumbing material supports persistence and proliferation of M. avium.     

Black Yeasts-Like Fungi Isolated from Dialysis Water in Hemodialysis Units

Hemodialysis in patients with chronic renal failure promotes the removal of toxic substances, water, and minerals from the body and often takes place in specialized clinics. Microbial contamination of dialysis fluid is a serious problem in therapy. One of the sources of contamination is the water used to prepare the dialysate. In Brazil, legislation regulating the microbiological quality of water for dialysis does not cover waterborne microbes such as Pseudomonas, mycobacteria, and fungi. The aim of the present study was to quantify, isolate, and identify fungi present in water systems in six hemodialysis units in Curitiba, Paraná state, Brazil. Fungi were analyzed by surface plating and membrane filtration. Isolates were identified by morphology, while the dematiaceous fungi were identified by sequencing the rDNA ITS region. It was found that 66 % of the samples presented fungi, while black fungi were present in 46 % of all samples. Twenty-eight isolates from treated water for dialysis and dialysate were identified by sequencing and were found to be Exophiala pisciphila, E. cancerae, E. equina, and Rhinocladiella similis. The presence of dematiaceous fungi may pose a risk for debilitated hospitalized patients.     

Antagonism between two natural enemies improves biological control of a coffee pest: The importance of dominance hierarchies

The relationship between two natural enemies of Coccus viridis (green coffee scale), an important hempiteran coffee pest was determined using a combination of experimental and observational approaches. Adult and larval forms of Azya orbigera, a coccinellid beetle predator were included on leaves of coffee plants with healthy scale populations resulting in lower proportions of scales infected with the second natural enemy, an entomopathogenic fungus (Lecanicillium lecanii). C. viridis populations on leaves where A. orbigera were excluded exhibited twice as much fungal infection by L. lecanii. In addition, field surveys of C. viridis populations on whole coffee plants corroborated experimental findings with eight times less fungal infection for coffee plants where A. orbigera was present than for plants where the predator was absent a month prior to surveys of L. lecanii. Despite a reduction in fungal infection in both the experiment and survey, the presence of the beetle reduced overall biological control of the pest only in the experiment where the receiver of the antagonism (L. lecanii) was more dominant in controlling C. viridis than the instigator of the antagonism (A. orbigera). In the survey, A. orbigera was dominant over L. lecanii, resulting in equal to greater levels of biological control depending on the degree to which A. orbigera was dominant over L. lecanii. Our results indicate that a negative relationship exists between A. orbigera and L. lecanii, but that contrary to expectations, this antagonism may in some cases improve overall biological control of the shared pest target.     

Molecular Analysis of Shower Curtain Biofilm Microbes

Households provide environments that encourage the formation of microbial communities, often as biofilms. Such biofilms constitute potential reservoirs for pathogens, particularly for immune-compromised individuals. One household environment that potentially accumulates microbial biofilms is that provided by vinyl shower curtains. Over time, vinyl shower curtains accumulate films, commonly referred to as “soap scum,” which microscopy reveals are constituted of lush microbial biofilms. To determine the kinds of microbes that constitute shower curtain biofilms and thereby to identify potential opportunistic pathogens, we conducted an analysis of rRNA genes obtained by PCR from four vinyl shower curtains from different households. Each of the shower curtain communities was highly complex. No sequence was identical to one in the databases, and no identical sequences were encountered in the different communities. However, the sequences generally represented similar phylogenetic kinds of organisms. Particularly abundant sequences represented members of the α-group of proteobacteria, mainly Sphingomonas spp. and Methylobacterium spp. Both of these genera are known to include opportunistic pathogens, and several of the sequences obtained from the environmental DNA samples were closely related to known pathogens. Such organisms have also been linked to biofilm formation associated with water reservoirs and conduits. In addition, the study detected many other kinds of organisms at lower abundances. These results show that shower curtains are a potential source of opportunistic pathogens associated with biofilms. Frequent cleaning or disposal of shower curtains is indicated, particularly in households with immune-compromised individuals.     

Exophiala macquariensis sp. nov., a cold adapted black yeast species recovered from a hydrocarbon contaminated sub-Antarctic soil

A new black yeast species, Exophiala macquariensis is described that is a member of the ascomycete family Herpotrichiellaceae, order Chaetothyriales. The genus Exophiala is comprised of opportunistic pathogens isolated from clinical specimens as well as species recovered from hydrocarbon contaminated environments. Several species have been reported to be able to degrade benzene, toluene, ethylbenzene and xylenes. Here, a novel species of Exophiala (CZ06) previously isolated from a Sub-Antarctic, Macquarie Island soil that was spiked with Special Antarctic Blend diesel fuel (SAB) is described. This isolate has the capacity of toluene biodegradation at cold temperatures. Multilocus sequence typing showed that this fungus was closely related to the pathogenic species Exophiala salmonis and Exophiala equina. With the capacity to utilise hydrocarbons as a sole carbon source at 10 °C, this fungus has great potential for future bioremediation applications.     

Microbial activity in drinking water-associated biofilms

Microbial biofilms from surfaces in contact with water may play a beneficial role in drinking water treatment as biological filters. However, detrimental effects such as biofouling (i.e., biocorrosion and water quality deterioration) may also occur. In this study microbiological processes and factors influencing the activity of bacteria in biofilms were investigated by conventional cultivation methods. The presence of bacteria belonging to different ecophysiological groups was assessed during drinking water treatment, in biofilms developed on concrete, steel and sand surfaces. Influences of the treatment process, type of immersed material and physico-chemical characteristics of raw/bulk water and biofilms upon the dynamics of bacterial communities were evaluated. Results revealed intense microbial activity in biofilms occurring in the drinking water treatment plant of Cluj. Ammonification, iron reduction and manganese oxidation were found to be the predominant processes. Multiple significant correlations were established between the evolution of biofilm bacteria and the physico-chemical parameters of raw/ bulk water. The type of immersed material proved to have no significant influence upon the evolution of microbial communities, but the treatment stage, suggesting that the processes applied restrict microbial growth not only in bulk fluid but in biofilms, too.     

Survival of Mycobacterium avium in Drinking Water Biofilms as Affected by Water Flow Velocity, Availability of Phosphorus, and Temperature

Mycobacterium avium is a potential pathogen occurring in drinking water systems. It is a slowly growing bacterium producing a thick cell wall containing mycolic acids, and it is known to resist chlorine better than many other microbes. Several studies have shown that pathogenic bacteria survive better in biofilms than in water. By using Propella biofilm reactors, we studied how factors generally influencing the growth of biofilms (flow rate, phosphorus concentration, and temperature) influence the survival of M. avium in drinking water biofilms. The growth of biofilms was followed by culture and DAPI (4′,6′-diamidino-2-phenylindole) staining, and concentrations of M. avium were determined by culture and fluorescence in situ hybridization methods. The spiked M. avium survived in biofilms for the 4-week study period without a dramatic decline in concentration. The addition of phosphorus (10 μg/liter) increased the number of heterotrophic bacteria in biofilms but decreased the culturability of M. avium. The reason for this result is probably that phosphorus increased competition with other microbes. An increase in flow velocity had no effect on the survival of M. avium, although it increased the growth of biofilms. A higher temperature (20°C versus 7°C) increased both the number of heterotrophic bacteria and the survival of M. avium in biofilms. In conclusion, the results show that in terms of affecting the survival of slowly growing M. avium in biofilms, temperature is a more important factor than the availability of nutrients like phosphorus.     

Effect of temperature and water activity on mycelia radial growth,conidial production and germination of Lecanicillium spp. isolates and their virulence against Trialeurodes vaporariorum on tomato plants

Biocontrol of the whitefly Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae) using entomopathogenic fungi has been a difficult challenge under greenhouse conditions. In order to select fungal isolates adapted to high temperature and extremely low moisture nine isolates of Lecanicillium lecanii (Zimmerman) Zare & W. Gams, L. attenuatum Zare & W. Gams and L. longisporum (Petch) Zare & W. Gams (Hypocreales: Clavicipitaceae) were evaluated. In vitro assays were performed to determine colony radial growth, conidial production and conidial germination in three water activity media (a_w = 0.97, 0.98 and 1.00) at 28 and 32 °C. Virulence of Lecanicillium spp. isolates was evaluated against third instar T. vaporariorum on tomato plants at 23 °C. Colony radial growth, conidial production and germination decreased with the reduction in water activity, while 32 °C was extremely detrimental for all fungal isolates. However, some isolates were able to grow and produce conidia at low water activity and high temperature. Additionally, mortality above 60 % was recorded for one of these isolates. Practical implementation of biocontrol of T. vaporariorum under greenhouse production systems should consider the selection of those Lecanicillium isolates that show tolerance to the adverse environmental conditions in greenhouses.     

Persistence and Viability of Lecanicillium lecanii in Chinese Agricultural Soil

The entomopathogenic fungus L. lecanii has been developed as biopesticides and used widely for biological control of several insects in agricultural practice. Due to the lack of isolation/count methods for L. lecanii in soil, the persistence of this fungus in soil appears to have attracted no attention. A selective medium and count method for L. lecanii in soil based on cetyl trimethyl ammonium bromide (CTAB) was developed, and then the persistence and viability of this fungus in soil were investigated under field conditions between 2012 and 2014. The results showed that the rate of recovery for L. lecanii in soil on the selective CTAB medium was satisfactory. The minimum CFUs for L. lecanii on the selective medium (0.5 g/L CTAB) was about 10² conidia/g soil. The L. lecanii density in soil declined quickly in the first month after inoculation with fungal conidia, kept stable for 6 to 10 months, and then decreased gradually until undetectable. L. lecanii could persist for at least 14 months in the agricultural soil of northern China. The colony growth, conidia yield and germination rate on plates, as well as the median lethal concentration or times (LC₅₀ or LT₅₀) to aphids, mycelium growth in aphids and sporulation on aphids of L. lecanii did not change significantly during the persistence in soil. In general, the count method developed here was a very useful tool for monitoring the dynamics of natural or introduced L. lecanii populations in soil, and the data on the persistence of L. lecanii in soil reported here were helpful for biological control and environmental risk assessment.     

Association of the Plant-beneficial Fungus Verticillium lecanii with Soybean Roots and Rhizosphere

Colonization of soybean roots by the biocontrol fungus Verticillium lecanii was studied in vitro and in situ. For in vitro experiments, V. lecanii was applied to soybean root tip explant cultures. Four weeks after inoculation, the fungus grew externally on at least half of the roots (all treatments combined), colonizing 31% to 71% of root length (treatment means). However, when a potato dextrose agar plug was available as a nutrient source for the fungus, root tips inoculated soon after transfer were not colonized by V. lecanii unless Heterodera glycines was present. Scanning electron microscopy of colonized roots from in vitro cultures revealed a close fungus-root association, including fungal penetration of root cells in some specimens. In the greenhouse, soybeans in sandy soil and in loamy sand soil were treated with V. lecanii applied in alginate prills. The fungus was detected at greater depths from the sandy soil than from the loamy sand soil treatment, but fungus population numbers were small and variable in both soils. Root box studies coupled with image processing analysis of the spatial distribution of V. lecanii in sandy soil supported these findings. Verticillium lecanii was detected randomly in the rhizosphere and soil of root boxes, and was rarely extensively distributed. These in vitro and in situ experiments indicate that V. lecanii can grow in association with soybean roots but is a poor colonizer of soybean rhizosphere in the soil environment.     

Candida albicans and Non-C. albicans Candida Species: Comparison of Biofilm Production and Metabolic Activity in Biofilms, and Putative Virulence Properties of Isolates from Hospital Environments and Infections

Candida albicans and, more recently, non-C. albicans Candida spp. are considered the most frequent fungi in hospitals. This study analyzed Candida spp. isolates and compared the frequency of different species, that is, C. albicans and non-C. albicans Candida spp., and the origins of isolates, that is, from hospital environments or infections. Yeast virulence factors were evaluated based on biofilm production and metabolic activity. Hemolysin production and the antifungal susceptibility profiles of isolates were also evaluated. Candida spp. were highly prevalent in samples collected from hospital environments, which may provide a reservoir for continuous infections with these yeasts. There were no differences in the biofilm productivity levels and metabolic activities of the environmental and clinical isolates, although the metabolic activities of non-C. albicans Candida spp. biofilms were greater than those of the C. albicans biofilms (p < 0.05). Clinical samples had higher hemolysin production (p < 0.05) and lower susceptibility to fluconazole (p < 0.05). Non-C. albicans Candida spp. predominated in samples collected from hospital environments and infections (p < 0.05). These species had a lower susceptibility to fluconazole and amphotericin B, and their biofilms had higher metabolic activities than those produced by C. albicans, which may explain the increased incidence of fungal infections with these yeasts during recent years.     

Benzimidazole tethered pyrrolo[3,4-b]quinoline with broad-spectrum activity against fungal pathogens

Fungal infections caused by Candida and Cryptococcus are particularly dangerous for immunocompromised individuals. In this study, we identified that benzimidazole fused pyrrolo[3,4-b]quinoline compounds have potent antifungal activity against several clinical isolates of pathogenic fungal strains. Specifically, the compound 6a did not show cytotoxicity against mammalian cells at a concentration that inhibits the growth of fungal strains. In addition, the compound 6a also significantly reduced the metabolic activity of fungal cells in the Candida albicans biofilms. Collectively, our results indicate that benzimidazole fused quinoline compounds have a potential to develop as an antifungal agents.     

Exoantigen tests for the immunoidentification of fungal cultures

Exoantigen tests for the immunoidentification of fungal pathogens are playing a new and significant role in the diagnostic laboratory. Properly performed and controlled exoantigen tests lead to rapid, accurate identification of cultures of many fungal pathogens. The tests are particularly valuable in identifying dimorphic pathogens that are difficult to convert or with atypical cultures. We review the value of exoantigen tests for identifying mycelial form fungi: Aspergillus spp. Blastomyces dermatitidis, Coccidioides immitis, Exophiala jeanselmei, Histoplasma spp., Paracoccidioides brasiliensis, Penicillium marneffei, Pseudallescheria boydii, Sporothrix schenckii, Wangiella dermatitidis and certain dermatophytes. We discuss procedures for performing the tests and sources of error.     

Microbial Biofilm Formation and Contamination of Dental-Unit Water Systems in General Dental Practice

Dental-unit water systems (DUWS) harbor bacterial biofilms, which may serve as a haven for pathogens. The aim of this study was to investigate the microbial load of water from DUWS in general dental practices and the biofouling of DUWS tubing. Water and tube samples were taken from 55 dental surgeries in southwestern England. Contamination was determined by viable counts on environmentally selective, clinically selective, and pathogen-selective media, and biofouling was determined by using microscopic and image analysis techniques. Microbial loading ranged from 500 to 10⁵ CFU · ml⁻¹; in 95% of DUWS water samples, it exceeded European Union drinking water guidelines and in 83% it exceeded American Dental Association DUWS standards. Among visible bacteria, 68% were viable by BacLight staining, but only 5% of this “viable by BacLight” fraction produced colonies on agar plates. Legionella pneumophila, Mycobacterium spp., Candida spp., and Pseudomonas spp. were detected in one, five, two, and nine different surgeries, respectively. Presumptive oral streptococci and Fusobacterium spp. were detected in four and one surgeries, respectively, suggesting back siphonage and failure of antiretraction devices. Hepatitis B virus was never detected. Decontamination strategies (5 of 55 surgeries) significantly reduced biofilm coverage but significantly increased microbial numbers in the water phase (in both cases, P < 0.05). Microbial loads were not significantly different in DUWS fed with soft, hard, deionized, or distilled water or in different DUWS (main, tank, or bottle fed). Microbiologically, no DUWS can be considered “cleaner” than others. DUWS deliver water to patients with microbial levels exceeding those considered safe for drinking water.     

The role of biofilms and protozoa in Legionella pathogenesis: implications for drinking water

Current models to study Legionella pathogenesis include the use of primary macrophages and monocyte cell lines, various free-living protozoan species and murine models of pneumonia. However, there are very few studies of Legionella spp. pathogenesis aimed at associating the role of biofilm colonization and parasitization of biofilm microbiota and release of virulent bacterial cell/vacuoles in drinking water distribution systems. Moreover, the implications of these environmental niches for drinking water exposure to pathogenic legionellae are poorly understood. This review summarizes the known mechanisms of Legionella spp. proliferation within Acanthamoeba and mammalian cells and advocates the use of the amoeba model to study Legionella pathogenicity because of their close association with Legionella spp. in the aquatic environment. The putative role of biofilms and amoebae in the proliferation, development and dissemination of potentially pathogenic Legionella spp. is also discussed. Elucidating the mechanisms of Legionella pathogenicity development in our drinking water systems will aid in elimination strategies and procedural designs for drinking water systems and in controlling exposure to Legionella spp. and similar pathogens.     

Subcutaneous Phaeohyphomycosis Caused by <Emphasis Type="Italic">Exophiala oligosperma</Emphasis> in an Immunocompetent Host: Case Report and Literature Review

We report a case of subcutaneous infection caused by Exophiala oligosperma. Erythematous ulcerated plaque with exudate was major clinical features. Histopathological examination showed yeast-like cells and fungal hyphae. Mycological and molecular identification revealed E. oligosperma as etiologic agent. Local debridement and oral itraconazole were effective. To the best of our knowledge, this is the first report of phaeohyphomycosis caused by E. oligosperma in mainland China. This report highlights the potential role of E. oligosperma as an emerging cause of infection in immunocompetent patients.     

Review of Campylobacter spp. in drinking and environmental waters

Consumption of contaminated drinking water is a significant cause of Campylobacter infections. Drinking water contamination is known to result from septic seepage and wastewater intrusion into non-disinfected sources of groundwater and occasionally from cross-connection into drinking water distribution systems. Wastewater effluents, farm animals and wild birds are the primary sources contributing human-infectious Campylobacters in environmental waters, impacting on recreational activities and drinking water sources. Culturing of Campylobacter entails time-consuming steps that often provide qualitative or semi-quantitative results. Viable but non-culturable forms due to environmental stress are not detected, and thus may result in false-negative assessments of Campylobacter risks from drinking and environmental waters. Molecular methods, especially quantitative PCR applications, are therefore important to use in the detection of environmental Campylobacter spp. Processing large volumes of water may be required to reach the desired sensitivity for either culture or molecular detection methods. In the future, applications of novel molecular techniques such as isothermal amplification and high-throughput sequencing applications are awaited to develop and become more affordable and practical in environmental Campylobacter research. The new technologies may change the knowledge on the prevalence and pathogenicity of the different Campylobacter species in the water environment.