Thursday, 16 April 2015

Exophiala jeanselmei




Exophiala jeanselmei -Mitosporic fungi; Hyphomycetes

Note:  With the advent of molecular testing, some sources are stating that Exophiala jeanselmei should more correctly be referred to as the Exophiala jeanselmei complex.  See explanation at the bottom of this post.

Also: Compare Exophiala jeanselmei with Exophiala dermatitidis elsewhere in this blog by clicking here.

Ecology:
Exophiala species can be found worldwide and may be isolated from soil, decaying wood and fresh water sources. 

Pathogenicity:
Exophiala jeanselmei may be an agent of mycetoma[i] and phaeohyphomycosis[ii] has also been implicated as an agent of back grain mycetoma and chromoblastomycosis[iii].  While occurring worldwide, some evidence suggests that infections, particularly neurotropic infections, may be more prevalent in East Asia.

Macroscopic Morphology:
Colonies are initially moist and yeast-like in texture; however they soon form a velvety surface due to the production of aerial mycelia.  Some isolated can remain yeast-like without further development.
The colony can be brownish-black to greenish-black with the reverse being black in colour.
Exophiala jeanselmei grows slowly, maturing within about 14 days at 25ᵒC to 30ᵒC.

Exophiala jeanselmei on SAB after incubation for 3 weeks at 30ᵒC.
While this isolate matured rather quickly, compared to the "up to 14 days" stated by some sources, the colony expanded outward slowly. (Nikon)

 Exophiala jeanselmei on SAB after incubation for ~3 weeks at 30ᵒC.
Heaped up, folded appearance.  (Nikon)
 
Microscopic Morphology:
Young cultures with budding yeast-like cells may eventually develop septate hyphae.  Hypha, initially hyaline (clear) become pale brown to olivaceous as the colony ages.  The conidiogenous annellophores (structure producing annelloconidia) are slender, sometimes branched, and narrow towards the apex (tip).  The smooth annelloconidia (2.5 – 6.0 µm X 1.2 – 2.5 µm) are oval or ellipsoidal in shape and if undisturbed, accumulate in clusters around the tip and upper sides of the annellide.  Potato Dextrose Agar (PDA) or Corn Meal Agar (CMA) may enhance conidia production, however all the photos on this post were taken from isolates grown on Sabouraud Dextrose Agar (SAB) with copious conidia produced.

Exophiala jeanselmei - mycelial growth adhering to small piece of agar from a slide culture after 4 days of growth.  (250X, LPCB, DMD-108)

Exophiala jeanselmei - small clusters or balls of conidia become evident, dispersed along the mycelia at this magnification. (250X, LPCB, Nikon)

Exophiala jeanselmei -Individual conida can now be made out at this magnification.
(400X, LPCB, DMD-108)

Exophiala jeanselmei -septate hyphae appear as if vacuolated.  Conidiogenous structures (annellophores) do not appear to be highly differentiated from the hyphae themselves and can be intercalary (along the hyphae) or terminal (at the end of the hyphae).
(1000X, LPCB, DMD-108)

Exophiala jeanselmei -as above, with typical accumulation of the oval to ellipsoidal annelloconidia around the top, and along the sides of the annellophore.
(1000X, LPCB, DMD-108)

Exophiala jeanselmei -again, intercalary, and terminal production of annelloconidia.
(1000X, LPCB, DMD-108)

Exophiala jeanselmei -and another.
(1000+10X, LPCB, DMD-108)

Exophiala jeanselmei -Both clusters of conidia and individual conidia can be seen at the tips of the annellophores.  (1000, LPCB, DMD-108)

Exophiala jeanselmei -Branched annellophore with (annello)conidia.
(1000+10X, LPCB, DMD-108)

Exophiala jeanselmei - and another...
(1000+10X, LPCB, DMD-108)

Exophiala jeanselmei - ditto, as above.
(1000+10X, LPCB, DMD-108)

Exophiala jeanselmei -branched conidiophore at various stages of conidia production.
(1000+10X, LPCB, DMD-108)

Exophiala jeanselmei -okay, too many photos of the same structures!!
(1000+10X, LPCB, DMD-108)

Exophiala jeanselmei -the annellophore appears undifferentiated from the hyphae from which it arises.  Nice photo showing the annelloconidia accumulated around the apex of the annellophore from which they were produced.  Conidia are single-celled with the darkly staining band across the center of the conidia not being a septum. Also visible on many of the conidia is the remnants of a fringe, the scar that remains from where they were attached to the conidiophore.  This represents a specific method of reproduction and fringe or scar (annellide) is why we specify the conidiophore as an annellophore, and the conidia as an annelloconidia.  (1000+10X, LPCB, DMD-108)
 
Exophiala jeanselmei -as the colony matures, the hypha become pigmented which gives the colony its distinctive colour. (1000X, LPCB, DMD-108)

Exophiala jeanselmei -as above, just another view,
(1000X, LPCB, DMD-108)

Exophiala jeanselmei -an interesting shot showing the annellophore rising upwards from it's parent hyphae with the annelloconidia clustered about the apex.  Almost three-dimensional.
(1000X, LPCB, DMD-108)

Exophiala jeanselmei -copious amounts of conidia are produced.
(1000X, LPCB, DMD-108)

Exophiala jeanselmei -as previously stated, the darkly staining band across the center of the annelloconidium is not a septum.  The single-celled annelloconidia have the fringe or scar which remains at one end of the cell which is why the conidia are specifically referred to as annelloconidia.
(1000+10X, LPCB, DMD-108)

Exophiala jeanselmei -conidium germinating.
(1000X, LPCB, DMD-108)

Exophiala jeanselmei -annelloconidia (1000+10X, LPCB, DMD-108)



[i] Mycetoma is a chronic, progressively destructive morbid inflammatory disease usually of the foot but any part of the body can be affected. Infection is most probably acquired by traumatic inoculation of certain fungi or ‎bacteria into the subcutaneous tissue. (WHO)

[ii] Phaeohyphomycosis is a heterogeneous group of mycotic infection caused by dematiaceous fungi whose morphologic characteristics in tissue include hyphae, yeast-like cells, or a combination of these. (Wikipedia)

[iii] Chromoblastomycosis is a chronic fungal infection of the skin and the subcutaneous tissue caused by traumatic inoculation of a specific group of dematiaceous fungi. (Medscape)

Note:  As with most fungi, speciation of the genus Exophiala is undergoing change brought about by molecular analysis.  Some sources are now referring to the ‘Exophiala jeanselmei complex’.
Isolates from the United States that in the past had been identified as E. jeanselmei based on morphologic and physiologic characteristic have been shown by molecular methods to be mostly two newly named species closely related to E.jeanslemei, i.e. Exophiala oligosperma and Exophiala xenobiotica.  A lower percentage were found to belong to several other species of Exophiala.  The precise species in the complex can only be determined by molecular testing.


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Friday, 27 March 2015

Acremonium species



Acremonium species -Hypocreaceae Family (obsolete name Cephalosporium spp.)

Ecology:
Acremonium species are yet another cosmopolitan fungus (ie. found just about everywhere) which can be isolated from soils as well as decaying plant material.  There are about one hundred recognized species of Acremonium.

Pathogenicity:
Acremonium species is associated with ‘white grain mycetoma’[i], an infection most commonly of the foot.  Acremonium has also been implicated in meningitis, endocarditis, endophthalmitis and corneal ulcers.  Onychomycosis or ‘tinea unguium’ (fungal infections of the nail) may also be caused by localized infection with Acremonium.  Disseminated infections are rare and may result from traumatic injury and may be more likely in immunocompromised hosts.

Macroscopic Morphology:
Acremonium exhibits moderately rapid growth, rather flat colonies that may be slightly raised in the center.  The colony has been described as glabrous to membrane-like initially, becoming powdery, cottony or even felt-like as it matures.  The colony may be white to cream in appearance or even yellowish to coral or pinkish in colour.  The reverse is pale, yellowish to pinkish in colour.

 Acremonium species -Sabouraud Dextrose Agar (SAB) incubated at 30˚C for 18 days. (Nikon)

  Acremonium species -Sabouraud Dextrose Agar (SAB) incubated at 30˚C for ~21 days. (Nikon)

Note -On the Challenges of Plate Photography:
This is the same species as above with a few more days of incubation.  The reason I posted both these photos is to show the difficulty I have in expressing the true colour (and often texture) of the fungus even when grown on the same media and under identical conditions.  The first photo was taken with the plate placed against a white background and the second against a black background.

The other challenge is lighting.  I take these photos for my own entertainment and education - for "fun" as the title of the blog states.  The acute care lab I work out of is interested only in a quick and accurate identification of fungi in clinical specimens.  Documentation by photography is not a concern and we have no professional set up for taking photographs.  I have rigged up my own apparatus for use as a camera stand for use within a biological safety cabinet (BSC) (see the post entitled 'Toys').  Photography of macroscopic plates is confined to the BSC as the lab could quickly become contaminated with spores if fungal plates were to be examined outside of the BSC.  Many fungi produce vast quantity of spores which can become airborne with only the slightest breeze.

I have little control over the lighting.  I often take a large number of photos using both white and black backgrounds.  I simply use a sheet of white paper which I have run through a photocopier without a "target".  In other words, I leave the cover of the photocopier open and "copy the air" which results in a sheet of paper covered with back toner on one side but white on the back.  I can flip this paper over to the black or white side as I wish and as the background paper may be contaminated with spores, I can discard it safely when finished.

I use the fluorescent tube lighting of the BSC itself, I have an incandescent source of lighting I can bring into the cabinet, and I can use the flash unit on the camera itself in order to manipulate lighting.  Light coloured fungi often show more detail when on a dark background and dematiacious fungi (darkly pigmented) stand out better on white backgrounds - but not always!  I try to avoid cast shadows.  I also find that the flash often produces such glare and reflection off of the petrie dish and agar surface that it obscures the true nature of the organism.  By taking a large number of photos on various backgrounds and with various combinations of the lighting I have available, I can usually find a photograph that reflects the true characteristics of the fungus I am attempting to document.

Microscopic Morphology:
Acremonium produces septate hyphae from which erect, unbranched and tapering phialides extend.  Most phialides (but not necessarily all) have a basal septum which delimits them from the hyphae proper.  Conidia are oblong (2–3 X 4–8 µm) are usually one-celled, however bicellular conidia may occur.  The conidia are produced and accumulate as balls (rarely as chains) at the apices of the phialides but they are fragile and easily disrupted.

Phialide: A specialized conidiogenous cell (conidiophore) that produces conidia in basipetal succession without increasing in length. (Mycology Online)


 Acremonium species - above is a little piece of agar which adhered to the slide culture microscope cover slip when removed from the agar block (See Post on Slide Cultures).  From this little flake of agar you can see phialide extending outwards with little "balls" of conidia attached at their apices. (LPCB, DMD-108, 250X)

Acremonium species - a collection of hypha run through the center of the photo from which you can see the phialide bearing conidia at their apices.  This is like "micro-botany" with the fungi as tiny plants, the 'seeds' (conicia) at the tips of the stems.  (LPCB, DMD-108, 400X)

Acremonium species - As above but at a slightly higher magnification.
(LPCB, DMD-108, 400+10X)

Acremonium species - the individual conidia can now be seen, gathered at the tapering tips of the phialides.  (LPCB, DMD-108, 1000X)

Acremonium species - not the greatest shot but try to picture the three 'balls' of conidia siting at the top of the phialides which are extending upwards, towards you, the viewer.  The phialides are blurred (out of focus) as they are out of the focal plane of the camera, lying beneath the balls of conidia, where they attach to the hyphae.  (LPCB, DMD-108, 1000X)

Acremonium species - The same as above but viewed more from the side.  The originating hyphae and tapering phialides are slightly out of the focal plane of the camera.  The conidia that were produced at the apex of the phialide remains undisturbed as a ball around the top.
(LPCB, DMD-108, 1000_10X)

Acremonium species - The previous photos show the phialides and conidia almost in three-dimensions.  In the photos that follow, they are lying fairly flat, making it easier to see the features.
(LPCB, Nikon, 400X)

Acremonium species - in the center of the photograph, there are two phialides, both with a collection of conidia at their apices, where they remain after being produced.
(LPCB, DMD-108, 1000X)

Acremonium species - another view of the tapering phialides extending from their parent hyphae where elongated, ellipsoidal conidia lay gathered.
(LPCB, DMD-108, 1000X)

Acremonium species - pretty much the same as in the above photo.  The phialide at the very top of the photo appears to have produced only one conidium.  Perhaps it is younger than the others.
(LPCB, DMD-108, 1000X)

Acremonium species - another photo of tapering phialides and the ellipsoidal conidia gathered around the apices.  (LPCB, DMD-108, 1000X)

Acremonium species - insert -one tapering phialide with individual conidia gathered at the apex.
(LPCB, DMD-108, 1000X)

Acremonium species - Two phialides, side by side producing large quantities of conidia.
(LPCB, DMD-108, 1000+10X)

Acremonium species - tapering phialide with single ellipsoidal conidia at the apex.  A slight collarette can be seen remaining around the apex.  Conida already produced almost seem to have fallen down and accumulated around the base of the phialide and hypha.
(LPCB, DMD-108, 1000+10X)

Acremonium species - as previously -another view of the tapering phialides and the balls of conidia adhering to the apex of the phialides where they were produced.
(LPCB, DMD-108, 1000+10X)

Acremonium species - As previously.
(LPCB, DMD-108, 1000X)

Acremonium species - a few barren phialides can be seen and the large mass of dispersed, primarily single celled conidia seen throughout.
(LPCB, DMD-108, 1000X)

Acremonium species may be confused with Verticillium and some isolates of Fusarium where macroconidia are not present.  Rate of growth and colony morphology differs from that of Acremonium species and may provide initial clues for differentiation.


[i] Mycetoma, or maduromycosis, is a slow-growing bacterial or fungal infection focused in one area of the body, usually the foot.  Approximately one month or more after the injury, a painless nodule forms under the skin surface.  The nodules develop into a tumor which produces sinuses to drain fluid. The fluid contains tiny grains, which may be a clue as to the type of organism is causing the infection.


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Monday, 23 February 2015

Ochroconis species



Ochroconis species (Hyphomycete)  Fungus

Ecology:
Ochroconis species are primarily soil saprobes (live on decaying vegetative matter), found in the soil worldwide.   As of 2014, there are thirteen recognized species of Ochroconis.

Pathology:
Ochroconis species have been recovered from central nervous system (CNS) infections as well as pulmonary (lung) infections, from both immunocompromised and immuocompetent hosts.  In particular, Ochroconis gallopava is considered to be a neurotropic opportunist and proposals have been made to place this fungus into a new genus, Verruconis. 
Ochroconis species are considered to be mesophilic (preferring moderate temperatures) however they can cause disease in several species of cold-blooded animals, particularly fish such as coho salmon and rainbow trout.  Ochroconis species are known to cause encephalitis in chickens, turkeys and other fowl.

Macroscopic Morphology:
The rate of growth is rather slow growing as measured by the expanding colony but will mature to produce conidia usually within 5 days.
The texture is described as velvety to felt-like or floccose.
The colony colour is usually a reddish-brown to chocolate brown to a dark olive-grey.  The reverse is a dark brown to black.
A red to brown pigment may diffuse into the medium.

 Ochroconis on Sabouraud Dextrose Agar (SAB) incubated at 30˚C for 3 weeks. (Nikon)

Ochroconis- same organism as above but with different background and lighting to show variations in texture and pigment.  SAB, 30˚C, 3 weeks. (Nikon)



Ochroconis on SAB - colony center rises off and above the agar surface resembling and inverted shallow bowl.  Looked like a small hollow mountain! 
Below, right - shows the Reverse of the Ochroconis presented here.  The lighter section which appears in the center of the larger colony is the area that has "cupped" and lifted off of the surface of the agar.  (Nikon

Microscopic Morphology:
Ochroconis produces septate hyphae which are hyaline (clear) to pale brown in colour.
Conidiophores are also hyaline to pale brown. They arise erect and unbranched from the hyphae and usually have a knobby or bent appearance.  The conidiophores have apical denticles in a sympodial arrangement from which the conidia have formed.  Conidia (2.5 – 4.5 µm X 11 – 18 µm) are usually 2 to 4 celled, depending on the species.  Conidia are cylindrical to club shaped and after detachment from the conidiophore (denticle), an inconspicuous frill may remain on both the denticle and the conidium base.

Ochroconis species - edge of growth of slide culture as initially viewed at low magnification.  Hyphae radiating out from point of inoculation after 1 week of incubation.
(LPCB, 250X, DMD-108)

Ochroconis species - Conidia extending from hyphae now become evident at this higher magnification. (LPCB, 250X, Nikon)

Ochroconis species - as we once more increase magnification individual conidia attached to phialides can be seen in more detail.  (LPCB, 400X, Nikon)

Ochroconis species - another view with conidia attached to their phialides. Brown pigmentation seen in lower right of photo.  (LPCB, 400X, Nikon)

Ochroconis species - one more view.  Remember, the fungus grows in three dimensions and even here, within the space between a microscope slide and a cover slip, hyphae and phialides extend forward, into the photo and some backwards, out of the photo.  It is for this reason that the photos often appear to be out of focus as only those features that lie relatively flat along the focal plane appear clearly in the picture. (LPCB, 500X, Nikon)

Ochroconis species - a massive ammount of conidia with the most mature to the right where the brown pigment is most evident. (LPCB, 500X, Nikon)

Ochroconis species - edge of  slide culture (as previous)
(LPCB, 500X, Nikon)

Ochroconis species - a hypha weaves its way from top center to bottom center of this photograph.  Along its length you can see phialides with attached conidia.  (LPCB, 500X, Nikon)

Ochroconis species -a two-celled conidium is seen attached to a phialide that extends from the hypha.  The conidium shows a slight constriction near the center.
(LPCB, 1000X, Nikon)

Ochroconis species -Numerous phialides with attached conidia shown here.  Phialides seen bearing multiple conidia.  Dark pigmentation is also evident as the colony ages.  Most conidia are two-celled, some showing a slight constriction near their center, others (center) showing the furthest end of the conidium being larger than that nearest the phialide from which it originated.  The arrow points to a phialide which has lost it's conidium -a slight scare remains.  
(LPCB, 1000X, DMD-108)

Ochroconis species -a mass of darkly pigmented, septate hyphae as well as a dark blue conidium seen near the left of the photo (LPCB, 1000X, DMD-108)

Ochroconis species -Hyphae with phialides bearing conidia.  Arrows point to phialies with multiple (two) conidia, the one on the left showing the scar remaining after the conidium has detatched.
(LPCB, 1000X, DMD-108)

Ochroconis species -a three-celled conidium appears to be present along with the more numerous two-celled conidia.  (LPCB, 1000+10X, DMD-108)

Ochroconis species -center of photo, conidium attached to a long phialide.
(LPCB, 1000X, DMD-108)

Ochroconis species -Nice photo of a phialide bearing two conidia attached to the parent hypha.
(LPCB, 100X, DMD-108)

Ochroconis species -yet another photo showing much the same.  Phialides bearing multiple conidia with the arrows showing the ragged attachment points which remain after the conida have detatched.
(LPCB, 1000+10X, DMD-108)

Ochroconis species -septation within the hyphae are clearly visible as is the developing brown pigmentation.  Tree Conidia still attached to their brownish pigmented phialides which extend from the hyphae.  Near center, one phialide is seen with a detached conidium nearby.
(LPCB, 1000X, DMD-108)

Ochroconis species -Aging phialides & conidia.  Brown phialide in center of photo has a thinner denticle at it's apex to which the conidium is attatched.
(LPCB, 1000X, DMD-108)

Ochroconis species -again, septations in hyphae are clearly evident.  Conidium near center of photo appears to be supported by a bent denticle.  (LPCB, 1000+10X, DMD-108)

Ochroconis species -conidium at apex attached to phialide by a short denticle.
(LPCB, 1000X, DMD-108)

Ochroconis species - Seen more clearly here, the two-celled conidium is attached to the phialide via a somewhat wavy denticle.  The phialide, in turn is attached to the hypha from which it originated.
(LPCB, 1000+10X, DMD-108)

Ochroconis species -  Here we are looking at the conidium 'head-on' so it appears spherical.  The conidium is attatched to the hypha by this darkly pigment, and apparently degenerating, phialide-denticle structure. (LPCB, 1000+10X, DMD-108)

Ochroconis species - Almost done here.  A phialide bearing two conidia.
(LPCB, 1000+10X, DMD-108)

Ochroconis species
(LPCB, 1000+10X, DMD-108) 

Ochroconis species - Phialide bearing multiple conidia.
(LPCB, 1000+10X, DMD-108) 

Physiology:
Growth is inhibited by cycloheximide.
I'm uncertain as to which specific species I have pictured in this blog as the conidia occasionally show more than 2 cells and can be constricted in the center.  The majority of the conidia from the isolate presented here are two-celled and rather ellipsoidal or cylindrical in shape


Differentiation of the more common species:
Conidia usually 4-celled = Ochroconis tshawytschae
Conidia usually 2-celled = Ochroconis gallopava
Conidia distinctly clavate (club-shaped), with the upper cell wider than the basal cell = Ochroconis humicola
Conidia broadly ellipsoidal and constricted at the septum = Ochroconis constricta

Ochroconis constricta is not known to be pathogenic
 
A new genus Verruconis is proposed for the neurotropic opportunist Ochroconis gallopava.

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