Cladophialophora species

Cladophialophora species  (Cladosporium species?) Fungus

Note:  This fungus was isolated as a plate contaminant and initially thought to be of the Cladosporium species however certain features discussed within this post left me to question the initial identification.  Recently, several of the more pathogenic species of Cladosporium have been reclassified under the genus Cladophialophora which this isolate appears to more closely resemble.  One of the more pathogenic species, Cladosporium bantiana was reclassified as Xylohypha bantiana and most recently as Cladophialophora bantiana.  Because of the serious pathogenicity of this particular species, most textbooks advise not making slide cultures of this fungus.  Unfortunately you really can’t identify what you have until you examine it by adhesive tape preparation or slide culture.  All preparations were carried out in a laminar flow biological safety cabinet and the organism was suspended in Lactophenol Cotton Blue of which the phenol component is lethal to fungi.  Utmost safety was observed in growing, working with and disposing of this and all isolates.

I remain uncertain of the genus/species of this fungus and certainly would welcome comments on the organism discussed below.

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Ecology: Both Cladosporium species and Cladophialophora species are cosmopolitan fungi, widely distributed in soils and decaying plant material.  Cladosporium species are often found as laboratory media contaminants as was this probable Cladophialophora species.

Macroscopic:  The colony was very slow growing at 30^oC and was approximately 2 cm in diameter after 14 days of growth.  The colony was had a velvety texture and was brownish-black in colouration.  The reverse was a rather nondescript black.  An important feature was that this isolate also grew well at 42^oC.

 Cladophialophora species on SAB grown at 30^oC for 14 days

Microscopic: Septate hyphae were observed which had a brown to black appearance.  Conidiophores did not appear significantly differentiated from the hyphae.  Moderately long chains of conidia extended from the conidiophores which also showed moderately frequent branching.  Both of these features are important in distinguishing the highly pathogenic Cladophialophora bantiana, other Cladophialophora species and the relatively non-pathogenic Cladosporium species.  Cladophialophora bantiana is described as having rather long, wavy and sparsely branched chains of conidia while other species (eg. Cladophialophora carrionii) show moderate branching with shorter conidial chain length.  Cladosporium species show the greatest amount of branching and have shorter conidial chain length. In this regard, the isolate shown here best fits the description of a Cladophialophora species other than bantiana.  The chains of Cladosporium are said to be rather fragile, easily liberating individual conidia.  Chains on the isolate presented in this post did not disarticulate easily but remained intact during manipulation.

 Cladophialophora species: First impression - edge of slide culture at low power

(~ 72hrs, LPCB, 250X, DMD-108)

Cladophialophora species -septate hyphae with branching.

(LPCB, Nikon 400X)

 Cladophialophora species (?) -Fairly extensive branching which is a feature of Cladosporium species.

(LPCB, 400X, DMD-108)

Cladophialophora species - Hyphae with poorly differentiated conidiophores bearing chains of eliptical (oval) conidia.  Note the smaller conidia at the tips of several chains.  The conidia are produced 'acropetally' (with the youngest conidia produced at the apex (tip), as opposed to being produced and extended at the base. Dark pigment showing through the LPCB stain on some of the chains of conidia.        (Note 100 µm bar at top right of photo -LPCB, 400X, DMD-108)

Unicellular conidia vary in size from about 2-5 µm to 4.5- 10 µm depending on the species.  The conidia of Cladosporium species are ellipsoid to round in shape.  Conidia of Cladophialophora bantiana and Cladophialophora carrionii are ellipsoid in shape while Cladophialophora emmonsii have a ‘bent’ appearance and Cladophialophora boppii are fairly round in shape.

  Cladophialophora species showing branched chains of oval conida extending from main hyphae.  Only a few free conidia are present (inset) and these show a hilus (H) where the conidia once were attached.  Darker pigment visible in the older released conidia.

(LPCB, 400X, DMD-108)

Cladosporium species also exhibit ‘shield cells’, named for their appearance (as in sword & ‘shield’- see diagram which follows below).  Conidia formed by Cladosporium species show a prominent black scar (hilum) at their point of attachment.  Both features are absent or at best questionable on the isolate discussed on this post.

Cladophialophora species - branched chains of oval conidia

(LPCB, 1000X, DMD-108)

Cladophialophora species - branched chains of elongated oval conida extending from hyphae.  Smaller conida at apex as the conidia develop acropetally (youngest at tip).  Convincing 'shield cells'  are not seen in any of the photos presented in this post which is evidence against it being a Cladosporium species.  Very few free conida are noted suggesting the chains are not easily disrupted, suggesting again that the isolate is a Cladophialophora rather than Cladosporium species.

(LPCB, 1000X, DMD-108)

 

Cladophialophora species or Cladosporium species? - the same isolate but here is the only cell I found in all my photos (too many to post) that somewhat resembles a shield cell (arrow).  The moderate to extensive branching does favour the Cladosporium species identification. 

(LPCB, 1000+10X, DMD-108) 

*Variable29% grow up to 40^oC

**Table borrowed & modified from Larone, 1989

About 86% of Cladosporium species can liquefy gelatin as well as grow in the presence of 15% NaCl which distinguishes this fungus from Cladophialophora species.

Cladophialophora bantiana tolerates cycloheximide, assimilates nitrate and produces urease.  The unknown isolate discussed here was not tested against cycloheximide.

Pathogenicity:  Cladosporium species are generally considered to be non-pathogenic, however, as with most organisms, immunocompromised individuals may be at greater risk at developing an infection.

Many species previously grouped under the Cladosporium genus have been recently reclassified as Cladophialophora species.  Cladophialophora now appears to encompass the pathogenic species.

1.      Cladosporium sp.– generally non-pathogenic

2.      Cladophialophora carionii – causes chromoblastomycosis

3.      Cladophialophora bantiana – causes cerebral phaeohyphomycosis (neurotropic, causing brain abscess.)  Infection is frequently fatal.  Has been isolated from pulmonary sources.  Highly infectious even in immunocompetent hosts.

4.      Cladophialophora emmonsii – rare cause of subcutaneous phaeohyphomycosis

?       Cladophialophora species (post isolate) – found as contaminant, pathogenicity unknown but interestingly this isolate grew well at 42^oC as does the pathogenic Cladophialophora bantiana.

In summary, the isolate presented here shows moderate to extensive branching which leans towards Cladosporium species as the identification.  However, the rather delicate structure with the absence of shield cells points more towards Cladophialophora species. Chains of conidia are rather long and not so easily disrupted which again adds evidence favouring Cladophialophora species. The clincher for me was that this isolate grew with ease at 42^oC, a temperature at which Cladosporium and most other Cladophialophora species are inhibited.  As such this isolate appears to resemble Cladophialophora species more than it does Cladosporium species and possibly the highly pathogenic Cladophialophora bantiana as it is the only one I'm familiar with that grows at 42^oC.   

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Trichophyton tonsurans

Trichophyton tonsurans (Fungus, Dermatophyte)

Pathogenicity and Distribution: Trichophyton tonsurans is a cosmopolitan (found worldwide) dermatophyte (a fungus of skin, nails or hair).  It is the etiologic agent most frequently implicated in ‘ringworm’ infections of the scalp (Tinea capitis) in America.  Causes an endothrix[i] infection of the hair (penetrates hair shaft to grow within).  T.tonsurans is anthropophilic (prefers humans to animals) however sources vary on its infectivity.  One prominent source[ii] suggests T.tonsurans is Zoophilic, but is frequently transmitted to man.  Others have speculated that equine strains may have mutated to become anthropophilic.  Infections are more commonly found in heavily populated urban regions

Macroscopic Morphology: T.tonserans may have highly variable colony morphology.  Surface growth may be white, beige, greyish or pale to sulphurous yellow, rose coloured to brownish.  The surface texture also can vary from velvety or powdery to suede-like, often with radial or concentric furrows.

 Trichopyton tonsurans: SAB agar at 30^o C after 14 days incubation, (Nikon)

Microscopic Morphology:  T.tonsurans produces septate hyphae.  The most prominent feature is the numerous microconidia formed along the hyphae or on short conidiophores which grow perpendicular to the originated hyphae.  The microconidia are sessile (attached by a ‘base’ rather than a ‘stalk’).  They can also be highly variable is shape ranging from pyriform (tear-drop) to clavate (club-like) to cylindrical and even larger round balloon-like forms.  Macroconidia are usually rare and also show variation in shape & size from cylindrical to cigar shaped (10 – 65 µm by 4 – 12 µm).  They are somewhat thick walled with a smooth surface and usually contain between 2 to 4 cells within each.  Terminal and intercalary chlamydospores may also be present, particularly in older cultures.  Another source[iii] has stated that spiral coils and arthroconidia may be present.

 Trichophyton tonsurans: Slide culture - First look at low power shows a mass of hyphae with microconidia visible on hyphae towards lower right of photograph.  (LPCB, DMD-108, X250)

 Trichophyton tonsurans: A closer look shows the microconidia growing along the hyphae.  This typical "birds on a wire" appearance is typical of Trichophyton species.  (LPCB, DMD-108, X400)

Trichophyton tonsurans:  Another look, as above. microconidia growing along hyphae.  Arrow shows a widening of the terminal end of a hyphal element, perhaps a young macroconidium or development of arthrospores?
  (LPCB, DMD-108, X400)

Trichophyton tonsurans: Microconida growing along the septate hyphae.

(LPCB, DMD-108, X1000)

 Trichophyton tonsurans: Another view showing the highly variable shape of the microconidia as well as an intercalary chlamydospore at the middle left of the photograph.

(LPCB, DMD-108, X1000)

Trichophyton tonsurans: Septate hyphae with microconida growing along its length.

(LPCB, DMD-108, X1000)

Trichophyton tonsurans:  Microconidia alongside septate hyphae with possible development of macroconidia at top left of photograph.  (LPCB, DMD-108, X1000)

Trichophyton tonsurans:  Again, microconidia along the length of a septate hyhal element.

(LPCB, DMD-108, X1000+10)

Trichophyton tonsurans:  Thickening of hyphal element - development of macroconida?  or arthrospores?  (LPCB, DMD-108, X1000)

Trichophyton tonsurans: two intercalary chlamydospores within the hyphae.

(LPCB, DMD-108, X1000+10)

Trichophyton tonsurans: Intercalary chlamydospore in an older culture.

(LPCB, DMD-108, Chlamydospore)

Trichophyton tonsurans:  Macroconidium - three visible divisions within.  T.tonsurans' macroconidia often show an irregular or wavy (undulating/ S-shaped) structure.

(LPCB, DMD-108, X1000+10)

Trichophyton tonsurans: Macroconidia in center of photograph.  Note micron bar at top of photo.

(LPCB, DMD-108, X1000)

Trichophyton tonsurans:  Stepping back a magnification, this photo shows the microconidia (MI) along the length of various hyphae, a possible macroconidium (MA) and what appears to be an intercalary chlamydiospore (CHL)  (LPCB, Nikon, X400)

Trichophyton tonsurans: (LPCB, DMD-108, X1000)

Trichophyton tonsurans:  Another photo just to get a better feel for the organism

(LPCB, DMD-108, X1000)

Trichophyton tonsurans:  Macroconidium -micron bar reads 20.23 µm.

(LPCB, DMD-108, X1000)

Physiological Tests:

·         Urease Positive

·         Hair perforation test –Negative (exceptions have been noted)

·         Usually causes alkalinisation of BCP-Milk solids-glucose media

·         Growth is stimulated by the addition of thiamine.

·         Growth at 37^oC

Definitive identification by molecular diagnosis is becoming more popular where available.

Differentiation:  The abundant microconidia of varying sizes and shapes assist in distinguishing this species from T.mentagrophytes and T.soudanense.  T.tonsurans response to thiamine also aids in this purpose.

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[i] Source states that hair perforation is variable – Guy St-Germain B.S., Richard Summerbell Ph.D. Star Publishing -further details to follow)

ⁱⁱ Atlas of Cinical Fungi, 2^nd edition: G.S. de Hoog, J. Guarro, J. Gené & M.J Figueras; Centraalbureau voor Shimmelcultures ‘ Universitat Rovira I Virgili, 2000: ISBN 90-70351-43-9

[iii] Medically Important Fungi – A Guide To Identification  5^th ed. Davise H. Larone, MT (ASCP), Ph.D., F(AAM),

ASM Press, Washington D.C.

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Exophiala dermatitidis

Exophiala dermatitidis (Wangiella dermatitidis) Fungus

To compare to Exophiala jeanselmei elsewhere in this blog, click here to redirect.

Note: The current name for this fungus is Exophiala dermatitidis, changed fairly recently from Wangiella dermatitidis & Phialophora dermatiditis earlier still). Molecular studies have shown it is related to Exophilia as a separate species and does not warrant its own genus (Wangiella) containing the single species (dermatitidis). Originally it was thought this organism differed from Exophiala by its ability to produce phialides from which the conidia are produced.

Ecology: Exophiala dermatitidis is a cosmopolitan (found almost everywhere) saprobe (lives of off dying/decomposing material). It can be isolated from soil, decaying wood, plants and from water.

Macroscopic Morphology: Shown here on Sabouraud Dextrose Agar (SAB or SDA) incubated at 30^oC, colonies are black to olive-black, often with greyish to brownish colours showing through, particularly at the periphery where the growth is younger. The brownish pigment may diffuse into the surrounding media.  It somewhat resembles molasses or perhaps dirty old motor oil. Reverse is much the same with a dark grey to iron-black colour. Growth is slow to moderate as the organism visible growth appears quickly but the colony is rather slow to expand and mature.  A distinguishing feature is the sticky, wet, mucoid growth this fungus initially produces.  A slimy string can be produced between the colony and an inoculating loop attempting to pick it up.  Some filamentous growth may occur on prolonged incubation or after several subcultures.  One source suggests that SAB media may encourage this.

Hints:  I didn’t have much luck with adhesive tape preparations with this ‘goo-y’ growth. It’s like trying to pick up a drop of motor oil with scotch tape!  Initially the fungus produced only yeast cells however some filamentous growth was finally visible at the center of the colony (oldest growth) after prolonged incubation (1 month).  Other sources suggest the velvety or filamentous growth may be first seen at the periphery.  The plate photo below is of a subculture incubated for about one month at 30^oC.  The majority of the photos shown below were taken of Exophiala dermatitidis growing on a slide culture, harvested after about 10 days of incubation at 30^oC.  I found this to be the most effective technique for viewing this organism. Hyphae are sparse but can be found growing out of the mass of yeast cells.

 Exophiala dermatitidis on SAB after about 1 Month at 30^oC

This organism grows fairly slowly and produces a wet, sticky, rather black looking colony.  Reverse (not pictured) is much the same.  Hyphal growth may appear after prolonged incubation and perhaps  is encouraged by repeated subculture.  Note the center of the growth appears more dry. The colony above was so shiny and glistening from the wet appearance that white bright spots on the left side of the colony are simply reflected light. (Nikon)

Microscopic Morphology:  Yeast cells are abundant with early growth appearing hyaline (clear) but acquire an olive colouration quickly. The sparse hyphae are septate and also have a pale olivaceous appearance in a wet mount. Conidiophores are poorly differentiated, often indistinguishable from the vegetative hyphae.  Phialides are also poorly differentiated and are generally ellipsoidal in shape.  Exophiala dermatitidis may somewhat resemble Phialophora however they do not produce the collarettes that are present on Phialophora.  The phialides can be intercalary (produced along the hyphae) or terminal (at the end).  Conidia are smooth, one-celled, subglobose to ellipsoidal (2.5 - 4 µm by 2 – 3 µm).  Somewhat spherical phialides may also be present producing phialoconidia of about the same size.  The conidia generally accumulate around the apex (tip) of the phialide.

 Exophiala dermatitidis - Not much detail here but this photo shows the yeast cells mass at the bottom of the photo from which hyphae have grown outwards. This was taken from the growth adhering to a microscope cover-slip taken from a slide culture after 10 days incubation at 30^oC.

(LPCB DMD-108 X40)

 Exophiala dermatitidis - as above (LPCB DMD-108 X250)

Just to give you an initial view of the colony before cranking up the magnification.

  Exophiala dermatitidis - at higher magnification the typical structures now appear.  At the bottom of the photo you can see the conidiogenous cells with conidia that have accumulated around their tip.  The cells at the top of the photo are out of the plane of focus but show much the same.

(LPCB DMD-108 X400)

Exophiala dermatitidis - Branched hyphae visible from which the poorly differentiated conidiogenous structures extend.  Oblong phialides can be seen and numerous oval conidia can be seen, particularly gathered around the somewhat obscured phialides on the left of the photo.

(LPCB, DMD-108 X1000)

 Exophiala dermatitidis- Elongated ovoid conidia seen gathered at the tip of one hyphal element and along it's length.  Inset & arrows: show the attachment point along the hyphae.

(LPCB, DMD-108 X1000)

 Exophiala dermatitidis - Shows hyphae which have poorly differentiated conidiogenous cells at the tip (ie. they don't look much different from the hyphae itself.  The phailides are the terminal cells from which the conidia are produced.  Again the conida can be seen accumulating around the tip of the cell from which they were produced.  Production of conidia can be seen at the end (terminal phialides) and along the length (intercalarly phialides).

(LPCB, DMD-108 X1000+10*)

 Exophiala dermatitidis- Hyphae seen with oval or 'vase' shaped intercalary phialides at various locations along it's length.  Again the far end of the hyphae shows poorly differentiated conidiogenous cells with a terminal phialide.  Conidia accumulate as clusters around both.

(LPCB, DMD-108 X1000)

  Exophiala dermatitidis- Another view shows hyphae with conidiogenous cells branching off and the somewhat oval of 'vase' shaped phialides at their apex (tip).  Elongated, oval conidia have accumulated around the tip.

(LPCB, DMD-108 X1000)

  Exophiala dermatitidis - Yeast cell mass at right of photo with single hyphae in center showing accumulation of oval conidia round the tip and along it's length.

(LPCB, DMD-108 X400+10*)

  Exophiala dermatitidis - Septa in hyphae are visible.  Phialide extending from hyphae is seen with conidia accumulated around the apex.

(LPCB, DMD-108 X1000+10*)

  Exophiala dermatitidis - Another view of a branch from a septate hyphae which can be seen differentiating towards the end where the conidia are accumulating  (ie. the cells are taper inwards where their are septa - at least two are visible)

(LPCB, DMD-108 X1000)

Exophiala dermatitidis - Mass of yeast cells seen at right of photo with two phialides seen extending from hyphae near center and the conidia they have produced.

(LPCB, DMD-108 X1000)

  Exophiala dermatitidis - Hyphae with phialides showing accumulated oval conidia around their tip.

(LPCB, DMD-108 X1000+10*)

  Exophiala dermatitidis - Another example as in the previous photo.  Compare this to Phialophora verrucosa.  No collerette is produced by Exophiala dermatitidis but can be found on Phialophora.

(LPCB, DMD-108 X1000+10*)

   Exophiala dermatitidis - Yeast cells on left with hyphae extending outwards.  One phialides along length and an other(s) at end where conidia have accumulated at tip.

(LPCB, DMD-108 X1000+10*)

  Exophiala dermatitidis - Another photo, this with micron bar for scale.  Both Terminal (T) and intercalary (I) phialides are seen with the characteristic accumulation of conidia.

(LPCB, DMD-108 X1000)

Exophiala dermatitidis - Intercalary phialides producing conidia.

(LPCB DMD-108 X1000)

Exophiala dermatitidis - Another example (I hate textbooks with only one small B&W print of the fungus in question - you really can't get a feel of the organism)

A single hyphae extending from mass of cells on right (look like both yeast cells and conidia) with conidia accumulating around tip and along side of hyphae.

(LPCB, DMD-108 X1000*) 

 Exophiala dermatitidis - Back to the beginning.  Just a photo of a wet-mount from a 48 hour culture incubated at 40^oC.  Only a few cells are beginning to develop the dark colour.

(Saline, DMD-108 X1000*)

Differentiation:  Exophiala dermatitidis does not assimilate potassium nitrate and grows at 40^oC which differentiates it from the related Exophiala jeanselmei. While both Exophiala dermatitidis and Phialophora verrucosa produces black colonies, the wet, sticky texture of E.dermatitidis cannot be mistaken for the dry, velvety P.verrucosa.  As mentioned earlier, the phialides of E.dermatitidis do not have collarettes as P.verrucosa does.

Pathology:  Has been implicated in subcutaneous and disseminated phaeohyphomycosis, particularly after traumatic implantation.  E.dermatitidis has been isolated from pulmonary specimens and may occasionally cause pneumonia.  Intervenious and catheter devices are thought to be a possible portal of entry.  Cystic fibrosis patients may be at increased risk of acquiring an infection with E.dermatitidis.  The organism also shows increased neurotropism (preferential infection of the central nervous system).

 * The DMD-108 refers to the Leica Digital Microimaging Device microscope.  This microscope is capable of adding X10 digital magnification to any objective magnification.  A +10 after any stated magnification indicates this feature was used.  Read more about this microscope here.

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