Friday, 5 October 2012

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 30oC 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 42oC.

 Cladophialophora species on SAB grown at 30oC 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 40oC
**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 42oC 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 42oC, 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 42oC.