Saksenaea vasiformis complex

Saksenaea vasiformis complex - Zygomycete

Note:  I had been debating with myself whether to post this organism at all as I have so few photos to share.  The photos I am posting show only the most basic characteristic features that would clue you in that this is a Zygomycete.  Still, it may be of interest to some….and, what else am I going to do with the photos?

Until recently, Saksenaea vasiformis was recognized as the sole species within this genus.  Currently, three species are recognized[i] and can be differentiated by genetic, morphological and physiological characteristics.  Now recognized are; S. vasiformis which produces mainly cylindrical sporagniospores with rounded ends, S. erythrospora which has large sporangiophores and sporangia, which produce biconcave ellipsoidal sporangiospores, and S. oblongiaspora, characterized by oblong sporangiospores and its failure to grow at 42ᵒC.

Ecology:  Saksenaea species can be found worldwide, having been isolated from soils in India, Brazil, Panama, Honduras, as well as the United States.

Pathology:  Though found worldwide, Infections occur most commonly in tropical and sub-tropical climates.  Saksenaea species have been implicated in both human and animal disease.  Unlike many of the emerging fungal diseases, it appears that Saksenaea infects immunocompetent hosts more readily than those with underlying, and usually predisposing conditions.  The most common mode of infection is through some traumatic implantation of material containing the sporangiospores though the respiratory tract may be the route of infection with disseminated infections.  Clinical symptoms and presentation can be quite varied –from a slow, localized invasion to rapidly spreading disseminated infection.  The spectrum of infections described in the literature range from skin and soft tissue infection to bone (osteomyelitis) and rhino-orbito-cerebral involvement.  Skin and soft tissue infections may present with necrotizing fasciitis or cellulitis.  While infections remain localized, they may respond to a combination of necrotic tissue debridement and aggressive antifungal therapy, otherwise amputation may be the only recourse.  Overall the mortality rate as a result of infection is about 40%[ii].  Disseminated infections have a significantly higher associated mortality rate of about 75%, while rhino-orbito-cerebral infections have an estimated mortality rate of 83% based on published reports.  Thankfully, infections with Saksenaea species remain relatively uncommon.

 Saksenaea species characteristics (in general) follow

Macroscopic Morphology:

A rapidly expanding fluffy, spider-like colony on Sabouraud Dextrose Agar incubated at 30ᵒC.  The growth may fill the Petrie dish within 48 hours.  It may be a “lid-lifter” and growth attempt to escape through poorly sealed plates after 72 plus hours.  The colony appears off-white to greyish in colour.

 Saksenaea species - 48 hours growth on SAB (or SDA) at 30ᵒC (Nikon)

Saksenaea species -as above, but an oblique view (Nikon)

Saksenaea species - 4 days growth on SAB (or SDA) at 30ᵒC (Nikon) 

Microscopic Morphology:

Saksenaea produces broad hyaline, mostly aseptate, hyphae.  Simple and unbranched sporangiophores (24 – 64 µm in length), develop a flask-shaped sporangium (50 – 150 µm in length).  The base of the sporangium above the hemispherical collumella is rather broad or swollen in appearance but narrows down into a long neck towards the apex.  Smooth walled sporangiospores (1.5 – 2.0 X 3 – 4 µm) vary in shape depending on the species as described above in ‘Notes’.  When mature, the sporangiospores are released through the top of the sporangiophore.  Dichotomously branching rhizoids develop at the base of the sporangiophore.  If this Zygomycete has produced its typical fruiting structure (sporangium), the overall structure of Saksenaea is unique and easy to identify.  But there lies the problem…

Problems:

It is rather difficult to induce Saksenaea to induce produce fruiting structures and subsequent sporulation.  Let’s back up a bit…

If you get a rapidly growing, fluffy colony which produces broad (wide) hyphae, there is a good chance you have a Zygomycete.  If you place the mould on relatively rich mycological media, such as Sabouraud Dextrose Agar or Potato Dextrose Agar, and don’t get fruiting structures produced, you may have either a Saksenaea species or Apophysomyces species.  Both are notoriously suborn when it comes to inducing sporulation on richer mycological media.  Media such as Czapek agar may produce better results.  If this fails and you have lots of patience, you can try the agar block – sterile water technique as outlined by Padhye & Ajello[iii].  While the experienced mycologist may be able to tell Apophysomyces from Saksenaea without inducing sporulation, it is valuable in further speciation when not resorting to other means of identification such as molecular.

Although I attempted the agar –sterile water technique described by Padhye & Ajello, I was not successful in inducing sporulation and therefore the distinctive flask-like sporangium was not produced, nor the spores within.  Features not produced cannot be photographed and I regret that I am unable to share them with you.  As I work in a clinical laboratory, we carry only basic media and time is limited –I regret that I could not continue to pursue this challenge.

Because the organism could not be induced to produce its fruiting structures and sporangiospores, it was impossible to use these morphological structures to determine the specific identification of this Saksenaea species.

 

Saksenaea species - so here is about all I could get.  The photo above shows the sporangiophore with the hemispherical shaped collumella at the apex (top).  From the inset photo you can see what the fungus would look like had it produced (or retained) the flask-like sporangium.

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

Important:  For the first time ever, I have posted a photo on this blog site which I have not personally taken.  I felt that I could not post the photos I had without showing what the mature and fully intact Saksenaea looked like.  The inset photo is shown here by the kind permission of Dr. David Ellis of the University of Adelaide's Mycology Website.  My photos are free to share, however I request that this photo not be shared as the inset photo is the property of Dr. Ellis.

Saksenaea species - line drawing of structures discussed

 Saksenaea species - again we see the hemispherically shaped collumella at the apex of the sporangiophore (arrows).  Sporangium and sporangiospores are absent.

(400X, LPCB, DMD-108) 

 Saksenaea species - as above

(400X, LPCB, DMD-108)

Saksenaea species - ditto

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

Saksenaea species - a clue that this is a zygomycete is the broad, almost aseptate hyphae that the mould produces.  The measurement seen within the large hyphae running through the photo reads 24.22 µm -that is wide!
  (400X, LPCB, DMD-108)

Saksenaea species - a close look at the hyphae in the previous photo.  Note that there is the septation at the center of the photo.  (1000+10X, LPCB, DMD-108)

 

 Saksenaea species - for the most part, the growth that I obtained, whether by slide culture or adhesive tape technique, appeared as above - just a tangled myceleum with no reproductive fruiting structures. (400X, LPCB, DMD-108)

So there you have it - the best I could do with the time and resources I had at hand.

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[i]Molecular phylogeny and proposal of two new species of the emerging pathogenic fungus Saksenaea

Alvarez, E, Garcia-Hermoso, D, Sutton, DA, Cano, JF, Stchigel, AM. Hoinard, D, Fothergill, AW, Rinaldi, MG, Dromer, F, Guarro, J.  Microbiol 2010;48 (12):4410-16

[ii] Mucormycosis caused by unusual mucormycetes, non-Rhizopus, -Mucor, and Lichtheimia species

Gomes, MX, Lewis, RE, Kontoyiannis, DP, Clin Microbiol Rev. 2011; 24(2):411-445

[iii] Simple Method of Inducing Sporulation in Apophysomyces elegans and Saksenaea vasiformis

Arvind A. Padhye and Libero Ajello; Journ Clin Micro, Sept 1988; 1861 - 63

*   *   *

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

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[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)