From A.S.M
Fig. 1
Three-month-old Mycobacterium ulcerans colonies growing on Middlebrook 7H10 agar supplemented with 10% oleic acid, albumin, dextrose, and catalase (OADC).
Scale bar = 1 mm
Fig. 2
Five-month-old Mycobacterium ulcerans colonies growing on
Middlebrook 7H10 agar supplemented with 10% OADC. Scale bar = 1 mm.
Macroscopic
examination reveals the presence of large and small Mycobacterium
ulcerans colonies after 3 (Fig. 1) and 5 months (Fig. 2) of growth at
30°C on Middlebrook 7H10 agar supplemented with 10% oleic acid,
albumin, dextrose, and catalase (OADC). The buff-colored M. ulcerans
colonies on supplemented Middlebrook 7H10 medium appear dry, waxy,
wrinkled, and rough with irregular edges. Buff-colored colonies
qualitatively indicate absence of mycolactone toxin production. Colonies
of M. ulcerans on OADC-supplemented Middlebrook 7H10 agar are variable
in size and have elevated, condensed centers which gradually flatten
toward the irregular, undulating periphery (Fig. 1 and 2). Compared to
the younger M. ulcerans colonies in Fig. 1, the mature M. ulcerans
colonies are slightly larger and raised, exhibiting more compacted,
dense colony growth (Fig. 2).
Methods
Prior
to imaging, cultures were inoculated onto Middlebrook 7H10 agar
supplemented with 10% OADC from frozen stock cultures stored at -70°C.
Plates were wrapped with parafilm and incubated at 30°C for 3 or 5
months. Images for Fig. 1 and 2 were taken on a Fisher Scientific
Stereomaster zoom microscope equipped with a 1.3 megapixel color digital
camera head for direct real-time viewing via a computer monitor.
Discussion
Mycobacterium sp.
are classified as either slow or rapid growers. By definition, slow
growers require more than 7 days to observe visible colonies on a solid
medium, while rapid growers form colonies within 7 days. Similar to Mycobacterium tuberculosis, M. ulcerans is a member of the slow-growing group of mycobacteria. However, M. ulcerans
is considered extremely slow-growing as cultures must be incubated for 6
to 8 weeks (or longer) under appropriate laboratory conditions prior to
forming distinct colonies. Figures 1 and 2 are images of mature M. ulcerans colonies that were grown for approximately 4 to 5 months, respectively. M. ulcerans
grows optimally on mycobacteriological media (e.g., Löwenstein-Jensen
medium, Middlebrook 7H10 medium, etc.) under the same conditions as M. tuberculosis (which generally forms colonies on solid media in 3 to 4 weeks), except the M. ulcerans optimal growth temperature is 30 to 32°C (9).
M. ulcerans,
the causative agent of a human disease known as Buruli ulcer, is an
environmental mycobacterium of which the natural reservoir is unknown
(2). Worldwide, Buruli ulcer is the third most common mycobacterial
disease of immunocompetent humans, after tuberculosis and leprosy (14).
Human transmission is believed to occur via skin transmission by direct
inoculation or an insect vector (10, 14).
Most individuals infected with M. ulcerans
initially develop a small, painless, preulcerative skin nodule with
larger areas of indurated skin and edema (13). As the disease
progresses over 1 to 2 months, the infected skin begins to ulcerate with
characteristic necrosis of the subcutaneous fatty tissues, deeply
undermined edges, and vascular blockage. Because
M. ulcerans is
a very slow-growing mycobacterium, more serious and advanced ulcerative
disease manifests over several months. The necrotic ulcers can lead
to: extensive skin loss; damage to nerves, blood vessels, and
appendages; and deformity and disability, particularly in children (13,
14). One study reported that 26% of patients with healed Buruli ulcers
suffered from chronic functional disability (8).
In contrast to other pathogenic mycobacteria, M. ulcerans
is an extracellular pathogen that produces a secreted toxin known as
mycolactone (4, 5, 12). Mycolactone has both cytotoxic (ability to
damage or kill certain types of human cells) and immunosuppressive
(reduces the activation or responsiveness of the human immune system)
properties and is most likely responsible for tissue necrosis observed
in patients, as injection of purified toxin into experimental animals
causes disease characteristics similar to Buruli ulcer (6). Although
secondary bacterial infections can further complicate the extensive
ulcerative lesions, death from an M. ulcerans infection is rare (7).
Currently,
no vaccine is available for the prevention of Buruli ulcer (11).
Although antibiotic treatment has been shown to be effective in vitro
and in animal models (1), success in the clinical environment has been
limited, especially in the case of advanced ulcerative disease.
Accordingly, surgical excision, combined with antibiotic therapy,
prevails as an accepted remedy for these difficult-to-treat infections
(3, 15).
