Dark-pigmented biodeteriogenic fungi in etruscan hypogeal tombs: New data on their culture-dependent diversity, favouring conditions, and resistance to biocidal treatments.

Subterranean Cultural Heritage sites are frequently subject to biological colonization due to the high levels of humidity, even in conditions of low irradiance and oligotrophy. Here microorganisms form complex communities that may be dangerous through mineral precipitation, through the softening of materials or causing frequent surface discolorations. A reduction of contamination's sources along with the control of microclimatic conditions and biocide treatments (overall performed with benzalkonium chloride) are necessary to reduce microbial growths. Dark discolorations have been recorded in the painted Etruscan tombs of Tarquinia, two of which have been analyzed to collect taxonomical, physiological, and ecological information. Eighteen dark-pigmented fungi were isolated among a wider culturable fraction: nine from blackening areas and nine from door sealings, a possible route of contamination. Isolates belonged to three major groups: Chaetothyriales, Capnodiales (Family Cladosporiaceae), and Acremonium-like fungi. Exophiala angulospora and Cyphellophora olivacea, a novelty for hypogea, were identified, while others need further investigations as possible new taxa. The metabolic skills of the detected species showed their potential dangerousness for the materials. Their tolerance to benzalkonium chloride-based products suggested a certain favouring effect through the decreasing competitiveness of less resistant species. The type of covering of the dromos may influence the risk of outer contamination. Fungal occurrence can be favoured by root penetration.

All Treatment Parameters Affect Environmental Surface Sanitation Efficacy, but Their Relative Importance Depends on the Microbial Target.

Environmental sanitation in food manufacturing plants promotes food safety and product microbial quality. However, the development of experimental models remains a challenge due to the complex nature of commercial cleaning processes, which include spraying water and sanitizer on equipment and structural surfaces within manufacturing space. Although simple in execution, the physical driving forces are difficult to simulate in a controlled laboratory environment. Here, we present a bench-scale bioreactor system which mimics the flow conditions in environmental sanitation programs. We applied computational fluid dynamic (CFD) simulations to obtain fluid flow parameters that better approximate and predict industrial outcomes. According to the CFD model, the local wall shear stress achieved on the target surface ranged from 0.015 to 5.00 Pa. Sanitation efficacy on six types of environmental surface materials (hydrophobicity, 57.59 to 88.61°; roughness, 2.2 to 11.9 µm) against two different microbial targets, the bacterial pathogen Listeria monocytogenes and Exophiala species spoilage fungi, were evaluated using the bench-scale bioreactor system. The relative reduction ranged from 0.0 to 0.82 for Exophiala spp., which corresponded to a 0.0 to 2.21 log CFU/coupon reduction, and the relative reduction ranged from 0.0 to 0.93 in L. monocytogenes which corresponded to a 0.0 to 6.19 log CFU/coupon reduction. Although most treatment parameters were considered statistically significant against either L. monocytogenes or Exophiala spp., contact time was ranked as the most important predictor for L. monocytogenes reduction. Shear stress contributed the most to Exophiala spp. removal on stainless steel and Buna-N rubber, while contact time was the most important factor on HDPE (high-density polyethylene), cement, and epoxy.IMPORTANCE Commercial food manufacturers commonly employ a single sanitation program that addresses both bacterial pathogen and fungal spoilage microbiota, despite the fact that the two microbial targets respond differently to various environmental sanitation conditions. Comparison of outcome-based clusters of treatment combinations may facilitate the development of compensatory sanitation regimes where longer contact time or greater force are applied so that lower sanitizer concentrations can be used. Determination of microbiological outcomes related to sanitation program efficacy against a panel of treatment conditions allows food processors to balance tradeoffs between quality and safety with cost and waste stream management, as appropriate for their facility.

Inherited CARD9 Deficiency in a Patient with Both Exophiala spinifera and Aspergillus nomius Severe Infections.

PURPOSE: Caspase-associated recruitment domain-9 (CARD9) deficiency is an inborn error of immunity that typically predisposes otherwise healthy patients to single fungal infections and the occurrence of multiple invasive fungal infections is rare. It has been described as the first known condition that predisposes to extrapulmonary Aspergillus infection with preserved lungs. We present a patient that expands the clinical variability of CARD9 deficiency. MATERIALS AND METHODS: Genetic analysis was performed by Sanger sequencing. Neutrophils and mononuclear phagocyte response to fungal stimulation were evaluated through luminol-enhanced chemiluminescence and whole blood production of the proinflammatory mediator interleukin (IL)-6, respectively. RESULTS: We report a 56-year-old Argentinean woman, whose invasive Exophiala spinifera infection at the age of 32 years was unexplained and reported in year 2004. At the age of 49 years, she presented with chronic pulmonary disease due to Aspergillus nomius. After partial improvement following treatment with caspofungin and posaconazole, right pulmonary bilobectomy was performed. Despite administration of multiple courses of antifungals, sustained clinical remission could not be achieved. We recently found that the patient's blood showed an impaired production of IL-6 when stimulated with zymosan. We also found that she is homozygous for a previously reported CARD9 loss-of-function mutation (Q289*). CONCLUSIONS: This is the first report of a patient with inherited CARD9 deficiency and chronic invasive pulmonary aspergillosis (IPA) due to A. nomius. Inherited CARD9 deficiency should be considered in otherwise healthy children and adults with one or more invasive fungal diseases.

Mating type (MAT) locus and possible sexuality of the opportunistic pathogen Exophiala dermatitidis.

Sexual reproduction among the black yeasts is generally limited to environmental saprobic species and is rarely observed among opportunists in humans. To date, a complete sexual cycle has not been observed in Exophiala dermatitidis. In this study, we aimed to gain insight into the reproductive mode of E. dermatitidis by characterizing its mating type (MAT) locus, conducting MAT screening of environmental and clinical isolates, examining the expression of the MAT genes and analyzing the virulence of the isolates of different mating types. Similar to other members of the Pezizomycotina, the E. dermatitidis genome harbors a high mobility group (HMG) domain gene (MAT1-2-1) in the vicinity of the SLA2 and APN2 genes. The MAT loci of 74 E. dermatitidis isolates (11 clinical and 63 environmental) were screened by PCR, and the surrounding region was amplified using long-range PCR. Sequencing of the ∼ 12-kb PCR product of a MAT1-1 isolate revealed an α-box gene (MAT1-1-1). The MAT1-1 idiomorph was 3544-bp long and harbored the MAT1-1-1 and MAT1-1-4 genes. The MAT1-2 idiomorph was longer, 3771-bp, and harbored only the MAT1-2-1 gene. This structure suggests a heterothallic reproduction mode. The distribution of MAT among 74 isolates was ∼ 1:1 with a MAT1-1:MAT1-2 ratio of 35:39. RT-PCR analysis indicated that the MAT genes are transcribed. No significant difference was detected in the virulence of isolates representing different mating types using a Galleria mellonella model (P > 0.05). Collectively, E. dermatitidis is the first opportunistic black yeast in which both MAT idiomorphs have been characterized. The occurrence of isolates bearing both idiomorphs, their approximately equal distribution, and the expression of the MAT genes suggest that E. dermatitidis might reproduce sexually.

In vitro interactions between IAP antagonist AT406 and azoles against planktonic cells and biofilms of pathogenic fungi Candida albicans and Exophiala dermatitidis.

In vitro interactions of AT406, a novel IAP antagonist, and azoles including itraconazole, voriconazole, and fluconazole against planktonic cells and biofilms of Candida albicans and Exophiala dermatitidis were assessed via broth microdilution checkerboard technique. AT406 alone exhibited limited antifungal activity. However, synergistic effect between AT406 and fluconazole was observed against both planktonic cells and biofilms of C. albicans, including one fluconazole-resistant strain. Moreover, synergism was also demonstrated between AT406 and itraconazole against both planktonic cells and biofilms of E. dermatitidis. No interaction was observed between AT406 and voriconazole. No antagonism was observed in all combinations.

Swim bladder mycosis in pretty tetra (Hemigrammus pulcher) caused by Exophiala pisciphila and Phaeophleospora hymenocallidicola, and experimental verification of pathogenicity.

Spontaneous invasive and chronic disseminated mycosis affected Hemigrammus pulcher kept in a public aquarium, and infection was manifested by inappetence, exophthalmia, erratic swimming, eroded scales, anaemia of the gills and abdominal distension. Internally, there was a grossly swollen swim bladder with a thickened wall filled with a dark mass. The body cavities contained a clear, light amber fluid and a swollen intestine which was full of a watery fluid containing small gas bubbles. Histopathology revealed a granulomatous inflammatory response with fungal hyphae in the lumen and wall of the swim bladder, hepatopancreas, spleen and kidneys with signs of nephrohydrosis. Exophiala pisciphila and Phaeophleospora hymenocallidicola were isolated from the swim bladder, abdominal cavity and gastrointestinal tract. The exogenous source of infection was probably the ample wooden decoration and plants inside the aquarium. Koch's postulates were fulfilled by re-isolation of both fungal species from fish artificially infected under laboratory conditions. As P. hymenocallidicola is less capable of defence against phagocytosis, E. pisciphila probably played a major role. Severe clinical manifestations with 100% mortality developed in two fish species infected by E. pisciphila. A significant increase in the plasma levels of amino acids was observed as a result of the activation of proteolysis.

Biofilm formation of the black yeast-like fungus Exophiala dermatitidis and its susceptibility to antiinfective agents.

Various fungi have the ability to colonize surfaces and to form biofilms. Fungal biofilm-associated infections are frequently refractory to targeted treatment because of resistance to antifungal drugs. One fungus that frequently colonises the respiratory tract of cystic fibrosis (CF) patients is the opportunistic black yeast-like fungus Exophiala dermatitidis. We investigated the biofilm-forming ability of E. dermatitidis and its susceptibility to various antiinfective agents and natural compounds. We tested 58 E. dermatitidis isolates with a biofilm assay based on crystal violet staining. In addition, we used three isolates to examine the antibiofilm activity of voriconazole, micafungin, colistin, farnesol, and the plant derivatives 1,2,3,4,6-penta-O-galloyl-b-D-glucopyranose (PGG) and epigallocatechin-3-gallate (EGCG) with an XTT reduction assay. We analysed the effect of the agents on cell to surface adhesion, biofilm formation, and the mature biofilm. The biofilms were also investigated by confocal laser scan microscopy. We found that E. dermatitidis builds biofilm in a strain-specific manner. Invasive E. dermatitidis isolates form most biomass in biofilm. The antiinfective agents and the natural compounds exhibited poor antibiofilm activity. The greatest impact of the compounds was detected when they were added prior cell adhesion. These findings suggest that prevention may be more effective than treatment of biofilm-associated E. dermatitidis infections.

Disseminated infection due to Exophiala pisciphila in Cardinal tetra, Paracheirodon axelrodi.

Cardinal tetra, Paracheirodon axelrodi (Schultz, 1956), kept in an ornamental tank, was found to be affected by severe invasive mycosis. Externally, the disease manifested as abdominal swelling, and internally, the anterior part of the intestine was extremely bloated with abundant dematiaceous septate hyphae and an accumulation of fluid. Histopathologically, a granulomatous inflammatory response was observed in the intestine wall, kidney and spleen. We assume that the mycotic agent was primarily deposited in the intestine and was then gradually disseminated to the other organs. DNA sequencing of ITS and LSU rDNA regions and phenotypic characterization were used for identification of the isolated fungus. The obtained data confirmed that the infection was caused by Exophiala pisciphila. The disease was subsequently reproduced in the carp fingerling using intramuscular and intraperitoneal injection of a spore suspension. The 13th day after intramuscular infection, a marked elevation of neutrophils was recorded in the peripheral blood; this involved a proliferation of band forms and segmented forms. As far as we know, this is the first report of infection due to E. pisciphila in Cardinal tetra.

Shared Physiological Traits of Exophiala Species in Cold-Blooded Vertebrates, as Opportunistic Black Yeasts.

Several species of the genus Exophiala are found as opportunistic pathogens on humans, while others cause infections in cold-blooded waterborne vertebrates. Opportunism of these fungi thus is likely to be multifactorial. Ecological traits [thermotolerance and pH tolerance, laccase activity, assimilation of mineral oil, and decolorization of Remazol Brilliant Blue R (RBBR)] were studied in a set of 40 strains of mesophilic Exophiala species focused on the salmonis-clade mainly containing waterborne species. Thermophilic species and waterborne species outside the salmonis-clade were included for comparison. Strains were able to tolerate a wide range of pHs, although optimal growth was observed between pH 4.0 and 5.5. All strains tested were laccase positive. Strains were able to grow in the presence of the compounds (mineral oil and RBBR) with some differences in assimilation patterns between strains tested and also were capable of degrading the main chromophore of RBBR. The study revealed that distantly related mesophilic species behave similarly, and no particular trend in evolutionary adaptation was observed.

Virulence markers of opportunistic black yeast in Exophiala.

The black yeast genus Exophiala is known to cause a wide variety of diseases in severely ill individuals but can also affect immunocompetent individuals. Virulence markers and other physiological parameters were tested in eight clinical and 218 environmental strains, with a specific focus on human-dominated habitats for the latter. Urease and catalase were consistently present in all samples; four strains expressed proteinase and three strains expressed DNase, whereas none of the strains showed phospholipase, haemolysis, or co-haemolysis activities. Biofilm formation was identified in 30 (13.8%) of the environmental isolates, particularly in strains from dishwashers, and was noted in only two (25%) of the clinical strains. These results indicate that virulence factors are inconsistently present in the investigated Exophiala species, suggesting opportunism rather than pathogenicity.

Proteome of tolerance fine-tuning in the human pathogen black yeast Exophiala dermatitidis.

UNLABELLED: The black yeast Exophiala dermatitidis is a worldwide distributed agent of primary and secondary diseases in both immunocompromised and healthy humans, with a high prevalence in human-made environments. Since thermo-tolerance has a crucial role in the fungus persistence in man-dominated habitat and in its pathogenicity, three incubation temperatures (37, 45, 1 °C) and two time spans (1 h, 1 week) were selected to simulate different environmental conditions and to investigate the effect of temperature on the proteome of E. dermatitidis CBS 525.76. Using a novel protocol for protein extraction from black yeasts, 2-D DIGE could be applied for characterization of changes in total protein spot abundance among the experimental conditions. A total of 32 variable proteins were identified by mass spectrometry. Data about protein functions, localization and pathways were also obtained. A typical stress response under non-optimal temperature could not be observed at the proteome level, whereas a reduction of the metabolic activity, mostly concerning processes as the general carbon metabolism, was detected after exposure to cold. These results suggest that a fine protein modulation takes place following temperature treatment and a repertoire of stable protein might be at the base of E. dermatitidis adaptation to altered growth conditions. SIGNIFICANCE: E. dermatitidis is a pathogenic black yeast causing neurotropic infections, systemic and subcutaneous disease in a wide range of hosts, including humans. The discovery of the fungus high prevalence in man-made habitats, including sauna facilities, drinking water and dishwashers, generated concern and raised questions about the infection route. In the present work - which is the first contribution on E. dermatitidis proteome - the effect of different temperature conditions on the fungus protein pattern have been analyzed by using a gel-based approach and the temperature responsive proteins have been identified. The absence of a typical stress response following the exposure to non-optimal temperature was detected at the proteome level, along with a general reduction of the metabolic activity after exposure to cold. These results suggest that a very fine regulation of the protein expression as well as adaptations involving a basic set of stable proteins may be at the base of E. dermatitidis enormous ecological plasticity, which plays a role in the fungus distribution, also enabling the transition from natural to human habitat and to the human host.

From Glacier to Sauna: RNA-Seq of the Human Pathogen Black Fungus Exophiala dermatitidis under Varying Temperature Conditions Exhibits Common and Novel Fungal Response.

Exophiala dermatitidis (Wangiella dermatitidis) belongs to the group of the so-called black yeasts. Thanks in part to its thick and strongly melanized cell walls, E. dermatitidis is extremely tolerant to various kinds of stress, including extreme pH, temperature and desiccation. E. dermatitidis is also the agent responsible for various severe illnesses in humans, such as pneumonia and keratitis, and might lead to fatal brain infections. Due to its association with the human environment, its poly-extremophilic lifestyle and its pathogenicity in humans, E. dermatitidis has become an important model organism. In this study we present the functional analysis of the transcriptional response of the fungus at 1°C and 45°C, in comparison with that at 37°C, for two different exposition times, i.e. 1 hour and 1 week. At 1°C, E. dermatitidis uses a large repertoire of tools to acclimatize, such as lipid membrane fluidization, trehalose production or cytoskeleton rearrangement, which allows the fungus to remain metabolically active. At 45°C, the fungus drifts into a replicative state and increases the activity of the Golgi apparatus. As a novel finding, our study provides evidence that, apart from the protein coding genes, non-coding RNAs, circular RNAs as well as fusion-transcripts are differentially regulated and that the function of the fusion-transcripts can be related to the corresponding temperature condition. This work establishes that E. dermatitidis adapts to its environment by modulating coding and non-coding gene transcription levels and through the regulation of chimeric and circular RNAs.

Black yeast diversity on creosoted railway sleepers changes with ambient climatic conditions.

The environmental isolation of opportunistic pathogenic black yeasts, which are responsible for a wide spectrum of human infections, is essential to understanding the ecology of clinical fungi. Extreme outdoor environments polluted with aromatic hydrocarbons support the growth of black yeasts in unlikely places, such as railway sleepers. However, there are limited data concerning the diversity of these fungi growing on polluted railway sleepers. In this investigation, we examined 845 railway sleeper samples, obtained from 11 Turkish cities representing altitudes from 25 to 1,893 m, and inoculated the samples onto mycological media for the isolation of black yeasts. Ninety-four samples (11.1 %) yielded positive results for black yeast, with creosoted oak sleepers having a significantly higher number of isolates than concrete sleepers (p < 0.05). Identification based on the ribosomal DNA (rDNA) internal transcribed spacer region revealed the highest prevalence of Exophiala phaeomuriformis, followed by Exophiala dermatitidis, Exophiala heteromorpha, Exophiala xenobiotica, and Exophiala crusticola. This study revealed that railway sleepers harboring black yeasts were predominantly (>75 %) populated with thermophilic species. We observed that altitude might have a significant effect on species diversity. Briefly, E. phaeomuriformis exhibited growth over a wide altitude range, from 30 to 1,893 m. In contrast, E. dermatitidis had a remarkable aversion to low altitudes and exhibited maximum growth at 1,285 m. In conclusion, we speculate that one can predict what species will be found on railway sleepers and their probability and that species diversity primarily depends on sleeper type and altitude height. We believe that this study can contribute new insights into the ecology of black yeasts on railway sleepers and the railway factors that influence their diversity.

Eumycetoma of the foot caused by Exophiala jeanselmei in a Guinean woman.

Eumycetomas are chronic infectious entities characterized by presence of mycotic grains in (sub-)cutaneous tissues, after accidental inoculation of an exogenous filamentous fungus in the skin. The lesions evolve towards painless pseudotumor of the soft parts. We report the original case of a Guinean woman exhibiting eumycetoma of the right foot. Both laboratory tests identified a dematiaceous fungus, Exophiala jeanselmei, as the responsible infectious agent. A medical treatment with voriconazole alone was sufficient to notice a substantial clinical improvement. This finding is unusual as E. jeanselmei is uncommon in Guinea-Conakry, and as optimal treatment rather associate antifungal azoles and surgical excision.

Adaptation of the black yeast Wangiella dermatitidis to ionizing radiation: molecular and cellular mechanisms.

Observations of enhanced growth of melanized fungi under low-dose ionizing radiation in the laboratory and in the damaged Chernobyl nuclear reactor suggest they have adapted the ability to survive or even benefit from exposure to ionizing radiation. However, the cellular and molecular mechanism of fungal responses to such radiation remains poorly understood. Using the black yeast Wangiella dermatitidis as a model, we confirmed that ionizing radiation enhanced cell growth by increasing cell division and cell size. Using RNA-seq technology, we compared the transcriptomic profiles of the wild type and the melanin-deficient wdpks1 mutant under irradiation and non-irradiation conditions. It was found that more than 3000 genes were differentially expressed when these two strains were constantly exposed to a low dose of ionizing radiation and that half were regulated at least two fold in either direction. Functional analysis indicated that many genes for amino acid and carbohydrate metabolism and cell cycle progression were down-regulated and that a number of antioxidant genes and genes affecting membrane fluidity were up-regulated in both irradiated strains. However, the expression of ribosomal biogenesis genes was significantly up-regulated in the irradiated wild-type strain but not in the irradiated wdpks1 mutant, implying that melanin might help to contribute radiation energy for protein translation. Furthermore, we demonstrated that long-term exposure to low doses of radiation significantly increased survivability of both the wild-type and the wdpks1 mutant, which was correlated with reduced levels of reactive oxygen species (ROS), increased production of carotenoid and induced expression of genes encoding translesion DNA synthesis. Our results represent the first functional genomic study of how melanized fungal cells respond to low dose ionizing radiation and provide clues for the identification of biological processes, molecular pathways and individual genes regulated by radiation.

Biodegradation of BTEX in a fungal biofilter: influence of operational parameters, effect of shock-loads and substrate stratification.

The effect of relative humidity (RH: 30% to >95%) of a gas-phase mixture composed of benzene, toluene, ethylbenzene and para-, meta- and ortho-xylenes (BTEX), inlet concentrations (0.2-12.6 g m(-3)), and empty bed residence times (EBRTs) (48-144 s) was tested in a fungi-dominant biofilter. A maximum elimination capacity (EC(max)) of 244.2 gBTEX m(-3) h(-1) was achieved at a total inlet loading rate (ILR(T)) of 371.2 gBTEXm(-3) h(-1) (RH: 65%). The transient-state response was tested by increasing the ILR(T), in two steps, from ~50 to 850 gm(-3) h(-1) and from ~50 to 320 g m(-3) h(-1), at a constant EBRT of 41.7s. Increasing the ILR(T) reduced the total BTEX removal efficiency (RE(T)) from >97% to 35%, and from >90% to 60% during medium and high shock-load, respectively. When subjected to short (4d) and long-term (7d) shut-down periods, the biofilter was able to recover high EC(max) of, respectively, 200 and 72 gBTEX m(-)3 h(-1) after resuming operation.

Exophiala sp. LHL08 reprograms Cucumis sativus to higher growth under abiotic stresses.

Endophytic fungi are potential sources of secondary metabolites; however, they are little known for phytohormones secretion and amelioration of plant growth under abiotic stresses. We isolated a novel endophyte from the roots of Cucumis sativus and identified it as a strain of Exophiala sp. by sequencing internal transcribed spacer/large subunit rDNA and phylogenetic analysis. Prior to identification, culture filtrate (CF) of Exophiala sp. has shown significant growth promotion of Waito-C [a gibberellins (GAs)-deficient mutant cultivar] and Dongjin-byeo (normal GAs biosynthesis cultivar) rice seedlings. CF analysis of Exophiala sp. showed the presence of physiologically active GAs (GA1, GA3, GA4 and GA7) and inactive GAs (GA5, GA8, GA9, GA12 and GA20). Exophiala sp. had higher GAs in its CF than wild-type strain of Gibberella fujikuroi except GA3. Influence of Exophiala sp. was assessed on cucumber plant's growth and endogenous abscisic acid (ABA), salicylic acid (SA) and bioactive GAs under salinity and drought stresses. Exophiala sp.-treated plants have shown significantly higher growth and rescued the host plants from stress promulgated water deficit, osmotic and cellular damage. The altered levels of stress-responsive ABA showed low level of stress confined to endophyte-applied plants than control. Elevated levels of SA and bioactive GAs (GA3 and GA4) in endophyte-associated plants suggest stress-modulating response toward salinity and drought. In conclusion, symbiotic relations between Exophiala and cucumber have reprogrammed the host plant growth under abiotic stresses, thus indicating a possible threshold role of endophytic fungi in stress alleviation. This study could be extended for improving agricultural productivity under extreme environmental conditions.

Elucidation of distribution patterns and possible infection routes of the neurotropic black yeast Exophiala dermatitidis using AFLP.

Distribution of populations of the opportunistic black yeast Exophiala dermatitidis was studied using AFLP. This fungus has been hypothesized to have a natural habitat in association with frugivorous birds and bats in the tropical rain forest, and to emerge in the human-dominated environment, where it occasionally causes human pulmonary or fatal disseminated and neurotropic disease. The hypothesis of its natural niche was investigated by comparing a set of 178 strains from natural and human-dominated environments in Thailand with a worldwide selection of 107 strains from the reference collection of the CBS Fungal Biodiversity Centre, comprising 75.7% clinical isolates. Many isolates had unique AFLP patterns and were too remote for confident comparison. Eight populations containing multiple isolates could be distinguished, enabling determination of geographic distributions of these populations. Some of the populations were confined to Thailand, while others occurred worldwide. The local populations from Thailand contained strains from natural and urban environments, suggesting an environmental jump of the fungus. Strains from human brain belonged to widely dispersed populations. In some cases cerebral isolates were identical to isolates from the human intestinal tract. The possibility of cerebral infection through intestinal translocation was thus not excluded.