First cultivation and characterization of Mycobacterium ulcerans from the environment

Abstract

Background: Mycobacterium ulcerans disease, or Buruli ulcer (BU), is an indolent, necrotizing infection of skin, subcutaneous tissue and, occasionally, bones. It is the third most common human mycobacteriosis worldwide, after tuberculosis and leprosy. There is evidence that M. ulcerans is an environmental pathogen transmitted to humans from aquatic niches; however, well-characterized pure cultures of M. ulcerans from the environment have never been reported. Here we present details of the isolation and characterization of an M. ulcerans strain (00-1441) obtained from an aquatic Hemiptera (common name Water Strider, Gerris sp.) from Benin.

Methodology/principal findings: One culture from a homogenate of a Gerris sp. in BACTEC became positive for IS2404, an insertion sequence with more than 200 copies in M. ulcerans. A pure culture of M. ulcerans 00-1441 was obtained on Löwenstein-Jensen medium after inoculation of BACTEC culture in mouse footpads followed by two other mouse footpad passages. The phenotypic characteristics of 00-1441 were identical to those of African M. ulcerans, including production of mycolactone A/B. The nucleotide sequence of the 5' end of 16S rRNA gene of 00-1441 was 100% identical to M. ulcerans and M. marinum, and the sequence of the 3' end was identical to that of the African type except for a single nucleotide substitution at position 1317. This mutation in M. ulcerans was recently discovered in BU patients living in the same geographic area. Various genotyping methods confirmed that strain 00-1441 has a profile identical to that of the predominant African type. Strain 00-1441 produced severe progressive infection and disease in mouse footpads with involvement of bone.

Conclusion: Strain 00-1441 represents the first genetically and phenotypically identified strain of M. ulcerans isolated in pure culture from the environment. This isolation supports the concept that the agent of BU is a human pathogen with an environmental niche.

Figure 1. Mass spectroscopic analysis of ASL of M. ulcerans 00-1441 showing the hydrolysis product of mycolactone A/B at m/z 447.3 (*).

The intact mycolactone can be identified at lower intensity at m/z 765.9 (**).

Figure 2. Mass spectroscopic analysis of the ASL of M. ulcerans 00-1441 showing the mycolactone A/B sodium adduct at m/z 765.6.

(In this case an ion trap was set to select for positive ions in the range m/z 755–775.)

Figure 3. MS/MS fragmentation pattern of the mycolactone A/B ion at m/z765.6.

The two main characteristic fragments are those of the macrolide ring m/z = 429.4 (*) and the side chain at m/z = 359.3 (**).

Figure 4. Cytotoxic activity of M. ulcerans 00-1441 to BMDM.

BMDM were infected with M. ulcerans 00-1441 at an MOI of 1∶1. Macrophages were photographed by phase-contrast microscopy at 4 h (A), 4 days (B), and 6 days (C) postinfection. Macrophage cell rounding, shrinkage and detachment are present at days 4 and 6 post-infection.

Figure 5. Footpad swelling and bacterial proliferation during infection with isolate 00-1441.

BALB/c mice were infected subcutaneously in the footpad with 5.2 log¹⁰ AFB of M. ulcerans 00-1441. The degree of pathologic changes was assessed by measurement of footpad swelling (▪) (n = 8). The proliferation of bacilli in the footpad was determined by counting AFB (open bars) of footpad homogenates (n = 5). Significant differences were performed using Student's t test (**, p≤0.01).

Figure 6. Histologic sections of mouse footpads infected with M. ulcerans 00-1441.

BALB/c (A–C) and NMRI (D) mice were infected subcutaneously with 5.4 log¹⁰ M. ulcerans 00-1441. At different time points the footpads were harvested and processed for histologic analysis. Dermal edema could be found by the second week of infection (A), along with a necrotic center surrounded by both chronic and acute inflammatory infiltrates (B) (hematoxylin-eosin stained sections). After 4 weeks of infection, bacilli were observed co-localized with cells (C) and in large clumps in the necrotic center of the lesion (D) (Ziehl-Neelsen stained sections).

Figure 7. Ziehl-Neelsen staining of bone tissue of mice infected with M. ulcerans 00-1441 (A, B and inset of D, arrows).

NMRI mice were infected subcutaneously in the footpad with 5.4 log¹⁰ M. ulcerans 00-1441. At 6 weeks post-infection, bacilli were found both in the bone tissue and in the bone marrow (A, B and inset in D, arrows). Hemotoxylin-eosin staining of C and D reveals extensive destruction of bone. Outlines of dead cortical bone spicules are seen in C and D (arrowheads). Marrow is necrotic and replaced by chronic inflammation.

Figure 8. An example of Afrotropical Gerridae: Limnogonus hypoleucus (Gerstaecker).

Photo: Jérome Constant, Department of Entomology, Royal Belgian Institute of Natural Sciences, Brussels, Belgium.

Figure 9. Main steps followed to cultivate M. ulcerans in pure culture from an aquatic insect (Gerris sp.).