Fun With Microbiology (What's Buggin' You?): Biverticillate

Penicillium citrinum

Ecology: Penicillium citrinum is a commonly occurring filamentous fungus with worldwide distribution. It has been isolated from a variety of sources including soils, decaying vegetation, foodstuffs (beans, coffee, cereals & spices) as well as a variety of indoor environments.

Pathology: While Penicillium species are generally regarded as laboratory contaminants, or at best, opportunists, a number of species have been implicated as being involved in the disease process. While Penicillium species may be isolated from clinical specimens, it is commonly believed that a true infection can only be established by histological demonstration of tissue invasion. With that in mind, Penicillium citrinum has been reported in mycotic keratitis (eye), lung infections (pneumonia), a single case of a urinary tract infection (UTI) and one of pericarditis. Their contribution to the disease process may be secondary an additional underlying illness. As with all fungi, immunocompromised individuals may be at greater risk of infection including those rarely considered as pathogenic.

Macroscopic Morphology: Penicillium citrinum exhibits moderately slow growth on Sabouraud-Dextrose agar (SAB) at 30ᵒC. Surface texture is velutinous (soft, velvety surface) to floccose (woolly tufts of soft “hairs”). The colonial growth appears radially sulcate (narrow, deep furrows or radial grooves –like spokes on a wheel). The mature colony has a central greyish-turquoise to greyish-orange colour with a white periphery (outer edge). Exudates (extrolites) are frequently produced which appear as drops of liquid upon the surface of the colony. These may appear clear, to pale yellow, to a reddish-brown in colour. Some strains may also produce a soluble pigment which can diffuse into the surrounding medium. The reverse is a pale yellow to a light yellow-brown. Colours and growth characteristics are, of course, media and strain dependent.

Penicillium citrinum-SAB, 14 days incubation at 30ᵒC (Nikon)

Note the drops of exudate (extrolites) which have formed on the surface.

Colour variation due to maturing of colony but also a difference in my lighting for photography.

Exudates (or Extrolites): Some fungi can produce exudates as a by-product of their growth, many of which can be collected for commercial use. Mycotoxins are by-products (secondary metabolites) which are potent poisons. Penicillium citrinum produces Citrinin, a nephrotoxic mycotoxin which derives its name from the fungus. It may also produce other extrolites such as tanzowaic acid A, quinolactacins, quinocitrinines, asteric acid and compactin.

Microscopic Morphology: Penicillium citrinum produces septate, hyaline (clear, not pigmented) hyphae. Smooth-walled conidiophores stipes are rather long (100 – 300 µm) and is biverticillate (see diagram at end of post). Metulae are 12 – 15 µm in length which are found in whorls of 3 – 5 divergent structures. Phialides are ampuliform (flask-shaped) and about 7 – 12 µm in length. Conidia (2.2 – 3.0 µm dia.) are globose to sub-globose (round to off-round) and are smooth or have a finely roughened surface. Conidia resist disruption and form rather long chains. These characteristics: the metulae longer than the phialides and the conidia being both spherical and produced in well-defined chains, are distinguishing features of Penicillium citrinum.

Penicillium citrinum- distinguishing features of Penicillium 'species' can already be made out at low magnification. (250X, LPCB, DMD-108)

Penicillium citrinum- distinguishing features of Penicillium 'species' much more evident at 400X.

Typical "fingers" made up of the metulae and phialide structures from which chains of conidia extend. (400X, LPCB, DMD-108)

Penicillium citrinum- a mass of overlapping fruiting structures with copious amounts of conidia.

(1000X, LPCB, DMD-108)

Penicillium citrinum- a little less congested in this photo. Conidiophores (stipes) seen from which extend the metulae and conidia producing phialides. Conidia are globose (round) to sub-globose (somewhat off-round) in shape, (1000X, LPCB, DMD-108)

Penicillium citrinum- long metulae and the somewhat shorter phialides are clearly distinguishable in this photograph. The conidia are generally smooth or can have a finely roughened surface.

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

Penicillium citrinum- another view.

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

Penicillium citrinum- exhibits biverticillate branching meaning that the conidiophore can branch and the metulae & phialides extend from these branches. Triverticillate would have the conidia branching and then the branches also branching to finally produce the metulae & phialide fruiting structures.

(1000X, LPCB, DMD-108)

Penicillium citrinum- Phialides are ampuliform (flask-shaped) and about 7 – 12 µm in length. Again, conidia (2.2 – 3.0 µm dia.) are globose to sub-globose (round to off-round) and are smooth or have a finely roughened surface. (1000+10X, LPCB, DMD-108)

Penicillium citrinum- Here we see the proportions of the metulae (M) and the 'flask-shaped' phialides (P) with the metulae being substantially longer than the phialides, The biverticillate structure is evident in this photo. (ie. each branch extending from the conidiophore (stipe), branches only once and then bears a fruiting structure consisting of the metulae and phialides.

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

Penicillium citrinum- another example.

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

Penicillium citrinum- a few more photos to finish up.

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

Penicillium citrinum- suitable for framing!

(1000X, LPCB, DMD-108)

Penicillium citrinum

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

Penicillium citrinum

(400X, LPCB, DMD-108)

Penicillium citrinum- another colony showing the exudate (extrolites) which accumulate on the colony surface after extended incubation. These metabolites may be potent poisonous mycotoxins or might have beneficial uses in industrial or pharmaceutical applications. (Nikon)

Physiology: The spores of Penicillium citrinum fail to germinate at 5ᵒC and may show restricted growth at 37ᵒC.

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Penicillium

Ah, the lowly genus Penicillium; a few words and photos…

The genus Penicillium has upwards of 300 identifiable species most of which are considered to be contaminants rather than pathogens when encountered in the clinical laboratory. The exception is Penicillium marneffei, which is unique amongst the Penicillium species as it can exhibit thermal dimorphism (ie. temperature dependent Yeast-like or filamentous forms). It is capable of causing cutaneous infections as well as invading the spleen, liver and bones. While P.marneffei is usually isolated from immunocompromised individuals, immunocompetent persons may also become infected. P.marneffei is most frequently found in South East Asia (South-eastern China, Myanmar, Laos & Thailand) and has been isolated from visitors to that region. While systemic infections with other species of Penicillium are unlikely, they have been recovered from respiratory & urinary tract specimens as well as from corneal scrapings. As with other fungi previously considered contaminants, Penicillium species should be opportunistic and capable of causing infection in the severely debilitated patient.

Many species of Penicillium produce mycotoxins or by-products which can be detrimental (eg citreoviridin, ochratoxin), while others beneficial (eg. mycophenolic acid, rubratoxins) [i]. Of course the antibiotic Penicillin is derived from the name Penicillium. It was in 1928 that Alexander Flemming observed that a culture of Penicillium rubens exuded a substance which inhibited certain bacteria – the birth of antibiotic therapy.

Ecology: This ubiquitous fungus, capable of tolerating a wide range of climates, is found predominantly in regions that have a temperate climate. Penicillium species can be found in soils, decaying vegetation, grains and food stuffs.

Macroscopic: Most species exhibit rapid growth and become fully mature in about 5 days. The surface appearance is usually described as velvety to powdery. The colony colour varies with the species but is usually a green, blue-green or grey-green, often with a white edge. Exudates of various colours may also form on the surface. The reverse usually a pale cream to yellow but may be a more intense reddish-brown. An exception is P.marneffei which can produce a red surface coloration that can diffuse into the medium.

Penicllium species on SAB media. Many species are rapid growing however the one pictured above was photographed at day 7 at 30^oC

Microscopic: Penicillium produces septate, hyaline hyphae, about 1.5 µm to 5 µm in diameter. Conidiophores can be simple or branched depending on the species. They are described as Monoverticillate (phialides extend directly from the hyphae), Biverticillate (phialides extend from branches which extend from the hyphae), or Terviticillate (phialides extend from structures called metulae, appearing as secondary branches, which extend from primary branches extending from the hyphae) –make sense? Phialides produce the conidia, together with the metulae and the branches can be considered the conidiophore. Phialides are, to a greater or lesser degree, flask or ampule shaped depending on the species. The phialides produce smooth or rough, single celled conidia (~2.5 µm to 5 µm) which extend as basipetal chains. Conidia can vary in shape from spherical to ovoid to fusiform, again dependant on the species. The entire structure, conidiophore and extending conidia resemble a ‘brush’ or penicillus (Latin), from which the genus name was derived.

Penicillium structures which are used in identification/classification of the species

Penicillium species - just one example of the many Penicillium species. Rather hefty phialides as compared to the delicate, tapering phialides of Paecilomyces species. Rough conidia produced by this species.

(LPCB, 1000X, DMD-108)

Penicillium species - got a great photo, but it does show the septate hyphae and conidiophores. This species appears to be biverticillate in structure.

(LPCB, 1000X, DMD-108)

Penicillium species (LPCB, 1000X, DMD-108)

Penicillium species - More of the same, just pretty pictures. Note micron bar in upper right of photo. (LPCB, 1000X, DMD-108)

Penicillium species - Phialides taper somewhat towards the apex where this species produces chains of rough-walled conidia.

(LPCB, 1000X, DMD-108)

Penicillium species can be differentiated from Paecilomyces species as the later has long, pointed apical extensions to the phialides. In my opinion, this gives Paecilomyces a somewhat more elegant or delicate appearance. The longer phialides of Paecilomyces appear to splay out, away from the conidiophore, much more than the rather straight phialides of Penicillium (a trident vs. pitchfork). Penicillium also bears some resemblance to Gliocladium species however Penicillium species produce chains of conidia while Gliocladium species’ conidia accumulate as a ball at the apex of the conidiophore. It differs from Scopulariopsis species by forming phialides.

Penicillium species - computer wallpaper (1024 X 768) when posted.

This was sent to me in an e-mail (I assume in the public domain) and thought I'd share this bit of "fun" with the readers.

Probably most of us, including microbiologists, attribute the discover of Penicillin to Sir Alexander Flemming - and rightly so. It was Flemming who first noticed the inhibitory action of a contaminating colony of Penicillium notatum against his culture of Staphylococcus aureus. While Flemming performed some initial experiments with the fungus in an attempt to isolate the active ingredient, he soon gave up on the endeavor. Flemming did, however, publish a paper on his findings which, some ten years later, found its way to a group of Oxford scientists who took up the challenge. It was this Oxford team, composed primarily of Howard Florey, Norman Heatley and Ernst Chain who through their ingenuity and perseverance, were responsible for having penicillin developed as a 'world changing' antibiotic. Flemming, Florey and Chain shared the Nobel prize for their discovery.

The story of the discovery and development of Penicillin can be found in a wonderfully readable book by Eric Lax entitled 'The Mold in Dr. Florey's Coat'. I highly recommend it for anyone who has further interest on the subject.