Apophysomyces elegans "complex"

Apophysomyces elegans complex (Zygomycetes) Order Mucorales

Note: As of this blog post, most textbooks still list Apophysomyces elegans as the sole species in this genus.  Recent studies [i] have demonstrated a high variability among the 5.8S rRNA gene sequences of clinical strains of A.elegans.  This study suggests that A.elegans is actually a complex composed of several (three) newly proposed species.  They have proposed the species names A.ossiformis, characterized by bone-shaped sporangiospores, A.trapeziformis, with trapezoid-shaped sporangiospores, and A.variabilis with variable-shaped sporangiospores.  A.elegans remains as the fourth species of the Apophysomyces complex.  Physiologically, A.elegans is able to assimilate the glycoside esculin, whereas the three newly proposed species failed to assimilate esculin.  Recent studies suggest that A.elegans may actually be a non-pathogenic environmental species, while the remaining three species are etiologically linked to clinical disease.  Further studies are warranted.

Ecology:

Although the ecology of Apophysomyces is rather poorly described, this fungus appears to have widespread distribution and has been isolated from soils and decaying vegetation in India, Australia, Southeast Asia and the United States.

Pathology:

Apophysomyces is an occasional agent of Zygomycosis.  Infection by Apophysomyces species differs from other Zygomycetes infections in two ways.  Firstly, it occurs more frequently in immunocompetent than immunocompromised individuals, whereas other Zygomycetes primarily infect hosts with weakened immune systems.  Secondly, Apophysomyces infections are acquired directly by traumatic implantation rather than by inhalation of spores which then progress to rhino-cerebral or other disseminated infections.

Apophysomyces infection may result in necrotizing cellulitis or fasciitis (flesh eating) requiring aggressive surgical debridement and antifungal therapy.  Despite immediate and aggressive intervention, the prognosis is often poor as once established the fungus can quickly spread to adjacent tissues and distant sites via the blood stream. 

In 2011, a tornado carved a path of destruction through the American town of Joplin Missouri killing 160 citizens.  Many others were received traumatic injuries and of those, there were there were thirteen cases of necrotizing cutaneous zygomycosis due to Apophysomyces trapeziformis, five of which were fatal.

While the descriptions of Apophysomyces in this blog is gathered from a variety of sources under the name of Apophysomyces elegans, the species presented photographically in this blog post is Apophysomyces variabilis.

Macroscopic Morphology:

As with other Zygomycetes, Apophysomyces exhibits very rapid growth, often filling the petrie dish with profusely woolly mycelia in two to three days.  Growth (SAB, 30ᵒC) initially appears white or off-white in colour but may acquire a slight brownish-grey colour as the colony ages.  The reverse is white to pale yellow.

 Apophomyces variabilis - on SAB, 48 Hours at 30ᵒC (Nikon)

 Apophomyces variabilis - on SAB, 48 Hours at 30ᵒC (Nikon)

Microscopic Morphology:

Note: The isolate presented here arrived in our laboratory as a proficiency testing challenge.  An initial direct tease mount revealed very broad, aseptate hyphae, suggesting Zygomycetes.  The isolate grew exceedingly well (SAB, 30ᵒC) yet failed to produce any fruiting structures.  The combined observations of broad aseptate hyphae and failure to produce fruiting structures on routine mycological media raised suspicions that this isolate belonged to either an Apophysomyces species, or was Saksenea vasiformis.

Both Apophysomyces species and Saksenea vasiformis are known to be notoriously resistant to efforts attempting to induce sporulation.  Study of the structures associated with sporulation greatly assists the identification of the Zygomycetes and as such, techniques have been developed in an attempt to induce the spore production[ii].   Unfortunately, our laboratory supports an acute care hospital and we lack the facilities for any experimentation outside of the routine clinical protocol.  (ie. purchased prepared media: no autoclave, media components, etc.)  However this blog is entitled “Fun With Microbiology” and I had “fun” improvising the ‘sterile water and yeast extract sporulation media’ outlined in endnote ii with some success.

Microscopic Morphology Continued:

Apophysomyces species produce broad based (up to 10+ µm dia.) hyaline (clear, non-pigmented) hyphae which are almost entirely aseptate.  Sporangiophores are quite long (up to 540 µm), unbranched and usually produced singly from aerial hyphae.  Sporangiophores may be attached to the hyphae with a prominent structure resembling the ‘foot cell’ found in Aspergillus species.  The apex of the sporangiophore widens to form a structure called an apophysis (hence the genus name) which may be distinctly bell-shaped or vase-shaped (20 -58 µm dia.) distinguishing it from that of other Zygomycetes.  Yet other sources describe the apophysis as champagne glass shaped or perhaps more common to other Zygomycetes, funnel-shaped.  The columella (18 – 28 µm) is hemispherical (half of a sphere) in shape.  Sporangiospores (5.4 – 8.0 X 4.0 – 5.7 µm) have been described as smooth and subspherical to cylindrical in shape.  Recall from the initial ‘Note’ at the start of this blog, the newly proposed species are molecularly distinct but also appear to produce distinctly shaped sporangiospores.  Rhizoids are produced and are generally located beneath or to the side of the sporangiophores.

An apology: as I lacked the facilities to properly induce sporulation in this fungus, these photos are as good as I'm going to get.

 Apophomyces variabilis - edge of tease mount showing single, well defined sporangiophore with attached sporangium. (250X, LPCB, Nikon)

 Apophomyces variabilis - direct tease moount.

(400X, LPCB, Nikon)

Apophomyces variabilis - apophysis without sporangium visible.  Several sporangiospores still visible in the bottom of the rather funnel-shaped apophysis.

(400X, LPCB, Nikon)

Apophomyces variabilis -again, from the direct tease mount showing a rather damaged apophysis remaining at the apex of the sporangiophore.

(400X, LPCB, Nikon)

Apophomyces variabilis - young sporangiophore at the tip of the sporangiophore.

(400+10X, LPCB, DMD-108)

Apophomyces variabilis -as above but appears to be some development of the sporangiospores within the sporangium.  (400+10X, LPCB, DMD-108)

Apophomyces variabilis -maturing sporangium with sporangiospores visible within.

(400+10X, LPCB, DMD-108)

Apophomyces variabilis -much the same.

(400+10X, LPCB, DMD-108)

Apophomyces variabilis - produces long, un-branched sporangiophores.  One unique characteristic described in Apophysomyces elegans is a dark area (arrow) often found on the sporangiophore slightly below the apophysis.  (400+10X, LPCB, DMD-108)

Apophomyces variabilis -two sporangiophores.  The one on the bottom might be described as champagne flute-like in shape and there is at least one sporangiospore present within.

(400+10X, LPCB, DMD-108)

Apophomyces variabilis -probably an immature sporangiospore.

(1000X, LPCB, DMD-108)

Apophomyces variabilis -two columellas (?) with the one at the top of the photo showing sporangiospores still clinging to the surface.  The sporangium appears to have

dehisced (dissolved, split open).  (1000+10X, LPCB, DMD-108)

Apophomyces variabilis -looking down on a sporagium with a number of sporangiospores still attached to the surface.  Average size of the sporangiospores along the longer axis averaged 5.22 µm.

(1000+10X, LPCB, DMD-108) 

Apophomyces variabilis -an immature sporangium.

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

Apophomyces variabilis -more of the same.  The large width of the zygomycete hyphae is evident here as a hypha runs through the photo.  (1000+10X, LPCB, DMD-108)

Apophomyces variabilis -again, the broad hyphae of zygomyces in general.  The dimension reads 10.74 µm.  (400X, LPCB, DMD-108)

Apophomyces variabilis -the apophysis looks distinctly bell-shaped in this microphotograph.  Sporangiospores appear to be present within the developing sporangium.

(400+10X, LPCB, DMD-108)

Apophomyces variabilis -to try to convince you of the bell-shaped apophysis.  Rather than a funnel or cone shape where each wall runs on one continuous angle, the walls here start on one angle, the abruptly flare out at a greater angle, somwhat resembling a bell.

(400X, LPCB, DMD-108)

Apophomyces variabilis -a rather shorter sporangiophore with a rhizoid seen at the top of the photo and apophysis and broken sporangium spewing sporangiospores at the bottom of the photo.

(400X, LPCB, DMD-108)

Apophomyces variabilis -dissolved or broken sporangium seen at center-right of photo, again releasing sporangiospores.  Large bubble at lower left of picture spoils this photo.

(400+10X, LPCB, DMD-108)

Apophomyces variabilis -champagne flute-like apophysis with few sporangiospores still clinging to the interior.  (1000+10X, LPCB, DMD-108)

Apophomyces variabilis -sporangium is missing with the apophysis remaining at the apex of the sporangiophore.  Not at the base of the sporangiophore (right side), the foot-cell is clearly evident and has picked up an intense blue colour from the LPCB.

(400+10X, LPCB, DMD-108)

Apophomyces variabilis -foot-cell of Apophysomyces which resembles the same feature more commonly found in Aspergillus species.

(1000X, LPCB, DMD-108)

Apophomyces variabilis -a sporangium breaking apart to release the sporangiospores within.  They really do look variable in shape in this photo, true to the species name, 'variabilis'.

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

Apophomyces variabilis -rhizoid, appears to have wrapped itself around a hyphal element.

(400X, LPCB, DMD-108)

Apophomyces variabilis -small rhizoid at center.

(400X, LPCB, DMD-108)

Apophomyces variabilis -okay, now that's a rhizoid! 

(1000X, LPCB, DMD-108)

Zygomycetes - some of the structures mentioned here and common to most zygomycetes

Physiology:

Apophysomyces, unlike other Zygomycetes, exhibits resistance to cycloheximide and therefore should grow on Mycosel® and Dermasel ® media.

Apophysomyces grows well at 30ᵒC, 37ᵒC & 42ᵒC

---

[i] Molecular phylogenetic diversity of emerging mucoralean fungus Apophysomyces:  Proposal of three new species.

Eduardo Alvearez, Alberto M. Stchigel, Josep Cano, Deanna A. Sutton, Annette W. Fothergill, Jagdish Chander, Valentina Salas, Michael G. Rinaldi and Josep Guarro

Rev. Iberoam Microbiol., 2010 27(2) pg. 80 - 89 (for purchase)

[ii] Simple Method of Inducing Sporulation by Apophysomyceselegans and Saksenaea vasiformis.

Arvind A. Padhye and Libero Ajello

Journ. Clin. Microbio., Sept. 1988, pg. 1861 – 1863.   (free PDF)                                                                 

Sporothrix schenckii Complex ("Revisited")

Sporothrix schenckii Complex –Hyphomycetes  (Dimorphic Fungus)

Note1: I first added a post entitled Sporothrix schenckii back in November of 2008 while bedbound, recovering from a serious injury.  I had just discovering ‘blogging’ and toyed with the idea of posting a few film photos that I had tucked away in a drawer – just for the fun of it.  The poor quality photo demanded it be upgraded once I decided to keep up my blog ‘Fun with Microbiology’.  Finally, here is the upgraded blog post, perhaps more accurately entitled Sporothrix schenckii complex Revisited.

Note2:  In recent years, gene sequencing studies have revealed that the species previously known as Sporothrix schenckii is actually composed of several species.  The currently accepted species names of the species which comprise the complex are as follows:  S.albicans (formerly Sporothrix pallida), S.brasiliensis, S.globosa, S.luriei, S.mexicana, and S.schenckii , ‘sensu strictu’.  As many clinical laboratories may not have routine access to molecular technology for specific speciation, this blog simply describes Sporothrix schenckii complex with macroscopic, microscopic and physiological features found in most current textbooks.

Ecology:

Sporothrix is a cosmopolitan (found just about everywhere) fungus which is commonly isolated from soil and decomposing plant matter.  Peat moss is a particularly well known source of Sporothrix.  It may also be found on living plants such as rose bushes leading to what has been termed ‘rose handler’s disease’ where the fungus gains entry to the host through thorny pricks.

Pathogenicity:

Sporothrix schenckii is the agent responsible for sporotrichosis, a chronic infection that most frequently begins as a skin puncture with introduction of the fungus into the subcutaneous tissues.  Eventually it will involve the lymph nodes and lymphatic channels that drain the infected area.  Implantation of the fungus is usually by puncture by items contaminated with plant material harbouring the fungus such as wood splinters, sphagnum moss, hay or thorns as previously mentioned.  Pulmonary (respiratory) infection may also develop in predisposed individuals after inhaling fungal spores.  Rare cases of disseminated Sporothrix infection with a fatal outcome have been reported.

Laboratory acquired infections have also been reported.

Sporothrix schenckii is a thermally dimorphic fungus, meaning it can take on one of two forms depending on the temperature it finds itself in.

·         At 25ᵒC exhibits the mould (filamentous) form, with a glabrous, moist texture.  Initially white or cream coloured, the colony may acquire a black colour with aging

·         At 37ᵒC exhibits a yeast-like form, with a creamy texture, cream to beige in colour.

Macroscopic Morphology:

The mould or filamentous phase in greater detail:

Growth is described by most sources s moderately rapid to rapid, with the colony becoming mature within 7 days.

The filamentous phase grown on SAB or PDA is generally cream coloured with some sources describing orange to orange-grey colouration.  As the fungus matures, a salt & peppery brown or black colour develops with the colony retaining a narrow whitish border.  Isolates may vary in their colour, some being dark/black from initial growth.  Stock cultures kept for long periods may lose their dark colour completely.

The reverse of darkly pigmented colonies is usually dark in the center with a progressively lighter periphery.

Sporothrix initially has a moist appearance but becomes wrinkled and leather to velvety in texture as it ages.

Sporothrix schenckii - SAB, 30ᵒC, 3 weeks incubation (Nikon)

The Yeast Phase:

The yeast phase is best induced by growing the fungus on Brain-Heart Infusion (BHI) agar at 37ᵒC, and observing after several generations (subcultures).   At 37ᵒC, Sporothrix exhibits a yeast-like form, with a creamy texture, cream to beige in colour which also may darken with aging.

(Yeast Phase Photo further below)

 

Microscopic Morphology:

Fillamentous Mould Phase:

Sporothrix produces narrow (1 – 2 µm dia.) hyaline, septate and branching hyphae.

Sporothrix produces two types of conidia:

·         Slender, tapering conidiophores arise at right angles from undifferentiated hyphae.   Hyaline conidia are produced at a small swelling at the conidiophores apex by sympodial growth resulting in a “rosette-like” appearance.  These conidia (2 – 3 X 3 – 6 µm) are tear-drop shaped to round in appearance and unless disturbed, remain attached to the conidiophore in young cultures via thread-like denticles. (Rosette)

·         Single thick-walled brown to black (dematiaceous) sessile conidia (2 – 4 µm dia.) can also be present, arising directly from the hyphae.

 Sporothrix schenckii -Initial look at the growth attached to a cover slip from a slide culture.

(100X, LPCB, DMD-108)

Sporothrix schenckii - A closer look where detail begins to emerge.

(400X, LPCB, DMD-108)

Sporothrix schenckii - ditto

(400X, LPCB, DMD-108)

Sporothrix schenckii - At yet a higher magnification, fine structures emerge. Conidia are easily seen at the tips of conidiophores.  At the center of this photo one such structure is seen in a 'rosette' arrangement, typical for this fungus.

(1000X, LPCB, DMD-108)

Sporothrix schenckii - hyphae bearing conidiophores with conidia being produced sympodially at the apex.  (1000X, LPCB, DMD-108)

Sporothrix schenckii - appearing somewhat in 3-D, the hypha is seen running across the photo with conidiophores extending at right angles from the hypha.  Conidia formation is is seen at different stages with the conidiophore at the right having accumulated numerous conidia.

(1000X, LPCB. DMD-108)

Sporothrix schenckii - again in "3-D" perspective, you can see a number of hyphae below, and out of the plane of focus of the camera, bearing conidiophores which appear to be rising upwards, towards the camera/viewer.  Conidia are present at the apex.

(1000X, LPCB, DMD-108)

Sporothrix schenckii -septate hyphae are show and two well defined conidiophores are shown with a 'rosette' of conidia at the apex.

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

Sporothrix schenckii -another look at the conidiophores with conidia accumulated at the apex (tips).  Fine thread-like denticles can be seen attaching the ellipsoid or tear drop shaped conidia to the conidiophore.

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

Sporothrix schenckii -yet another view, as above.

(1000+10X. LPCB, DMD-108)

Sporothrix schenckii -what is meant as a "rosette" configuration of conidia.  This is typical of Sporothrix schenckii and assists in its identification.

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

Sporothrix schenckii - I particularly like this photo.  Like a shower of delicate flowers.  What is seen are the conidia rosettes at the tips of unseen conidiophores and hyphae, below the plane of focus.  (1000X, LPCB, DMD-108)

Sporothrix schenckii -a single hypha running through the photo, bearing conidia in typical rosette pattern.  (1000X, LPCB, DMD-108)

Sporothrix schenckii -single, sessile and begining to show dark pigment (dematiaceous), are seen along the hypae.  (1000X, LPCB, DMD-108)

Sporothrix schenckii -ditto.  Black pigment much more pronounced in this photo.

(1000X, LPCB, DMD-108)

Sporothrix schenckii - again, as above.

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

Sporothrix schenckii - just another photo...

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

Sporothrix schenckii -Sessile (attached directly to hypha) with many developing a dark pigmentation (dematiaceous) which gives the macroscopic colony its distinctive colour.

(1000X, LPCB, DMD-108)

Sporothrix schenckii - dematiaceous cells are more abundantly produced as the colony ages.

(1000X, LPCB, DMD-108)

Sporothrix schenckii -sessile dematiaceous conidia are seen lining a hypha running through the photo, as well as some conidiophores bearing the tear-drop shaped conidia.

(1000X, LPCB, DMD-108)

Sporothrix schenckii -another view of the sessile, dematiaceous conidia lining the hypha with a few conidiophores extending from the hypha showing the delicate rosette arrangement at the tips.  (1000X, LPCB, DMD-108)

Sporothrix schenckii -and just another photo showing what was described in the last few photos.

(1000X, LPCB, DMD-108)

Sporothrix schenckii -a nice rosette formation in the center of the photo.

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

The Yeast Phase:

As previously mentioned, the yeast phase is best induced by growing the fungus on Brain-Heart Infusion (BHI) agar at 37ᵒC, and observing after several generations (subcultures).  Yeast cells are round to ovoid of varying size (1 – 3 X 3 – 10 µm), producing single or multiple buds often resembling rabbit ears or ‘Mickey Mouse’ ears extending from the primary yeast cell.

 Sporothrix schenckii -the very same organism added to the nutritionally rich Brain-Heart Infusion (BHI) agar and incubated at 37ᵒC for ten days.  A typical pasty, yeast form develops which is why this organism is 'dimorphic'. (Nikon)

Sporothrix schenckii -yeast cells seen in a suspension in Lactophenol Cotton Blue.

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

Sporothrix schenckii - Sporothrix in the yeast phase may show single yeas like cells with some of the population budding with typical "rabbit ears" or "Mickey Mouse" ears.  They appear as two elongated cells projecting from the same surface of the parent cell.  One can be seen here, one-third in from the center left edge of the photo.  (1000X, LPCB, DMD-108)

Sporothrix schenckii -"Mickey Mouse' ear configuration in the cell at the upper center.

(1000X, LPCB, Nikon)

Sporothrix schenckii -again, yeast cells with two attached daughter cells on the same side giving the apearance of "rabbit ears" or "Mickey Mouse" ears.

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

Sporothrix schenckii -ditto (as above) -inset -floppy 'rabbit ears"

(1000X, LPCB, DMD-108)

Sporothrix schenckii - that's it, I'm done!

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

Physiology:

Sporothrix is resistant to cycloheximide; however growth is inhibited at temperatures of 39 - 40ᵒC.

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