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Morphology of Mature Mycobacterium ulcerans Colonies Send Print

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Created: Thursday, 28 April 2011
Last update: Monday, 14 November 2011
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Three-month-old Mycobacterium ulcerans

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.

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Three-month-old Mycobacterium ulcerans (Labeled view)

FIG. 1. Three-month-old Mycobacterium ulcerans colonies (labeled view).

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Five-month-old Mycobacterium ulcerans

FIG. 2. Five-month-old Mycobacterium ulcerans colonies growing on Middlebrook 7H10 agar supplemented with 10% OADC. Scale bar = 1 mm.

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Five-month-old Mycobacterium ulcerans (Labeled view)

FIG. 2. Five-month-old Mycobacterium ulcerans colonies (labeled view).

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Introduction

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).

References

1.    Dega, H., A. Bentoucha, J. Robert, V. Jarlier, and J. Grosset. 2002. Bactericidal activity of rifampin-amikacin against Mycobacterium ulcerans in mice. Antimicrob. Agents Chemother. 46:3193–3196.
2.    Edelstein, H. 1994. Mycobacterium marinum skin infections. Report of 31 cases and review of the literature. Arch. Intern. Med. 154:1359–1364.
3.    Etuaful, S., B. Carbonnelle, J. Grosset, S. Lucas, C. Horsfield, R. Phillips, M. Evans, D. Ofori-Adjei, E. Klustse, J. Owusu-Boateng, G. Amedofu, P. Awuah, E. Ampadu, G. Amofah, K. Asiedu, and M. Wansbrough-Jones. 2005. Efficacy of the combination rifampin-streptomycin in preventing growth of Mycobacterium ulcerans in early lesions of Buruli ulcer in humans. Antimicrob. Agents Chemother. 49:3182–3186.
4.    George, K. M., L. P. Barker, D. M. Welty, and P. L. Small. 1998. Partial purification and characterization of biological effects of a lipid toxin produced by Mycobacterium ulcerans. Infect. Immun. 66:587–593.
5.    George, K. M., D. Chatterjee, G. Gunawardana, D. Welty, J. Hayman, R. Lee, and P. L. Small. 2002. Mycolactone: a polyketide toxin from Mycobacterium ulcerans required for virulence. Science 283:854–857.
6.    George, K. M., L. Pascopella, D. M. Welty, and P. L. Small. 2000. Mycobacterium ulcerans toxin, mycolactone, causes apoptosis in guinea pig ulcers and tissue culture cells. Infect. Immun. 68:877–883.
7.    Johnson, P. D. R., T. P. Stinear, and J. A. Hayman. 1999. Mycobacterium ulcerans—a mini-review. J. Med. Microbiol. 48:511–513.
8.    Marston, B. J., M. O. Diallo, C. R. Horsburgh, Jr., I. Diomande, M. Z. Saki, J. M. Kanga, G. Patrice, H. B. Lipman, S. M. Ostroff, and R. C. Good. 1995. Emergence of Buruli ulcer disease in the Daloa region of Cote d'Ivoire. Am. J. Trop. Med. Hyg. 52:219–224.
9.    Portaels, F., P. Johnson, and M. W. Meyers. 2001. Buruli ulcer: diagnosis of Mycobacterium ulcerans disease. World Health Organization, Geneva, Switzerland.
10.    Portaels, F., and W. M. Meyers. 1999. Insects in the transmission of Mycobacterium ulcerans infection. Lancet 353:986.
11.    Tanghe, A., P. Adnet, T. Gartner, and K. Huygen. 2007. A booster vaccination with Mycobacterium bovis BCG does not increase the protective effect of the vaccine against experimental Mycobacterium ulcerans infection in mice. Infect. Immun. 75:2642–2644.
12.    van der Werf, T. S., T. Stinear, Y. Stienstra, W. T. van der Graaf, and P. L. Small. 2003. Mycolactones and Mycobacterium ulcerans disease. Lancet 362:1062–1064.
13.    van der Werf, T. S., W. T. van der Graaf, J. W. Tappero, and K. Asiedu. 1999. Mycobacterium ulcerans infection. Lancet 354:1013–1018.
14.    Weir, E. 2002. Buruli ulcer: the third most common mycobacterial infection. Can. Med. Assoc. J. 166:1691.
15.    World Health Organization. 2004. Provisional guidance on the role of specific antibiotics in the management of Mycobacterium ulcerans disease (Buruli ulcer). World Health Organization, Geneva, Switzerland.

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