Ross et al.
variant genome and therefore screened a genomic library of DNA fragments after digestion with AluI and HaeIII. Southern blot hybridization using a
genomic probe revealed a band of approximately 1.1 kb that was highly reactive in AluI-digested DNA, which appeared to be repeated at least 50 times in the
variant genome and therefore was considered as an ideal target for the PCR-based test
. This PCR test (primer pair MU1 and MU2, amplify between tandem copies of IS
. These authors showed that the length of the complete repeat element was 1274 bp, flanked by 7-bp direct repeats with 12-bp terminal inverted repeats and a single large open reading frame potentially encoding a protein composed of 328 amino acids
. Their results suggested that this repeat element may constitute an insertion sequence (IS) element, which is commonly found in other mycobacteria and usually used as a template for PCR amplifications
. This repeat was named IS
(amplified with primer pairs MU5 and MU6). They also discovered another insertion element of 1404 bp in length with 12-bp terminal inverted repeats, repeated between 30–40 times per genome, with a single open reading frame potentially encoding a protein of 445 amino acids that they named IS
. IS
appeared to be unrelated to and distinct from other known mycobacterial IS, such as IS
found in M. avium
found in M. gordonae
. At that time, specificity tests of the IS
was also present in the genome of M. lentiflavum which is not phylogenetically related to
The IS-based PCR tests, particularly IS
2404, have become the molecular basis for the detection of the
M. ulcerans variant from which more recent studies have attempted to improve their specificity and sensitivity
[28], notably through the implementation of the quantitative real-time PCR (qPCR) combined with the use of TaqMan probes that allow (1) confirmation of the identity of a DNA amplification product and (2) simultaneous amplification of multiple DNA targets
[29][30].
3. Implication for the Diagnosis and Treatment of BU
3.1. Implication for BU Diagnosis
BU has a wide spectrum of clinical manifestations
[31], with extremely aggressive infections in some areas while others are indolent or rapidly healing
[6]. These observations have raised the hypothesis that if, in a given area,
M. ulcerans is genotypically homogeneous, then host factors such as immunity, metabolism and nutrition may play an important role in the history of infection
[6]. However, while MPM variants were initially given different species names based on slightly different phenotypic characteristics, they all show great genotypic similarities making it clear that they represent rather variants of the same MPM complex. This means that a variety of MPM variants are capable of causing similar skin ulcerations all named BU, a scenario that could also explain the spectrum of clinical observations. This hypothesis is notably supported by the association of clinically diagnosed BU cases with
M. liflandii or M. liflandii-like variants
[16][32] or
M. marinum variants
[33]. In these cases, IS
2404 and KR-B PCR were both positive. While some studies reported negative cultures for the
M. liflandii variant
[32] others described positive cultures with phenotypic features similar to the
M. ulcerans variant such as slow growth (>6 weeks), yellowish coloration and granular appearance of the colonies
[16]. Then, whilst
M. liflandii and
M. pseudoshottsii variant infections have been mainly reported in frogs and fish, respectively, these germs may be under-detected in human lesions and should be looked for in order to determine their pathogenicity. Nevertheless, it is also important to keep in mind that although
M. marinum variant infections can be mistaken for BU, they usually present specific clinical syndromes such as verrucous plaques or sporotrichoid lesions which are not typical of
M. ulcerans variant infections
[34]. It is also noteworthy that even in lesions mimicking stricto sensu BU,
M. marinum variant lesions do not present undermined edges, which are the most specific sign of
M. ulcerans variant infections. Therefore, it is necessary to bear in mind that some strains of
M. marinum can also be responsible for a different nosological entity (the so-called “fish-tank disease”). Also, whilst
M. shinshuense and
M. ulcerans variant infections are characterized by similar clinical presentations, an aquatic exposure does not seem to be relevant in
M. shinshuense variant transmission
[35].
Has BU been underestimated? Since clinical diagnosis is usually based on careful examination of the ulceration and then confirmed either by IS
2404 PCR amplification and/or direct microscopy and/or histopathology (including AFB staining, a characteristic shared by all mycobacteria;
[36]) and/or culture, it seems that the answer is no. However, the genetic diversity of the mycobacterium capable of causing BU or BU-like disease has been underestimated.
3.2. Implication for M. ulcerans Genetic Diversity
Another question remains: do these MPM variants represent the genetic diversity of
M. ulcerans? Some authors have suggested that the
M. ulcerans variant has recently passed through an evolutionary bottleneck and adapted to a new niche environment
[10]. Tobias et al.
[9] referred to “niche-adapted” mycobacteria, with
M. liflandii and
M. ulcerans variants representing thus different ecotypes. Interestingly, it found both variants in the same environmental habitats (sediments) in French Guiana as well as in BU biopsies
[16], suggesting that these MPM variants share the same environments. Since BU is known to be caused by the
M. ulcerans variant worldwide, scholars propose to consider that all other variants belonging to the MPM complex represent
M. ulcerans’ variability. Nevertheless, scholars do not exclude that within this MPM complex the
M. ulcerans variant did not appear first but rather evolved from another ancestor variant. Notably, based on the number of ISs repeats (IS
2404 and IS
2606) and the number of pseudogenes present in the MPM genomes, scholars suggest that they could have evolved from an ancestor variant to adapt to the host they infect rather than to an ecological niche.
3.3. What about the Pathogenicity of Other Mycobacterium Species
Another important finding is the discovery of other mycobacterial species (not belonging to the MPM complex) associated with diagnosed BU cases. Stinear et al.
[19] showed that out of 50 clinical samples diagnosed as BU, only 40 were positives for IS
2404, raising the question about the etiology of the others. In Côte d’Ivoire,
M. chelonae and
M. smegmatis have been found in BU lesions
[15] and more recently based on metabarcoding analysis of clinical samples,
M. gilvum has been found associated with some BU cases in French Guiana
[17]. For the latter cases, AFB (BAAR) staining was negative, IS
2404 and KR-B qPCR were both negative, and the culture on LJ solid medium was negative
[17]. As it has shown that
M. gilvum does not harbor IS
2404, IS
2606 and KR-B in its genome and plasmids (
Figure 1), this raises questions about its mechanisms of pathogenicity in humans. Taken together, these results raise the possibility that other mycobacterial species may have other types of virulence genes and immunosuppressive properties as the MPM variants that enable them to cause skin ulcerations similar to those caused by MPM variants. Furthermore, these results highlight the potential of metabarcoding studies, usually used for environmental investigations, in the search for the infectious agent responsible for human disease. These broader molecular screens could, for example, help the medical community target the most appropriate treatment and thus avoid late responses to inappropriate antibiotic therapy.
3.4. Implication for BU Treatment
The World Health Organization (WHO) recommends a combination of rifampicin (10 mg/kg once daily) and clarithromycin (7.5 mg/kg twice daily) to treat BU
[37]. In Australia, although not proven effective, a combination of rifampicin (10 mg/kg once daily) and moxifloxacin or ciprofloxacin (400 mg once daily) is routinely used to treat patients (WHO, 2022). In Japan, a triple combination of rifampicin, levofloxacin and clarithromycin is used
[38] while in French Guiana, treatment consists of a combination of rifampicin, amikacin or clarithromycin
[39]. Whilst all based on rifampicin, BU treatment also combines other antimicrobial molecules that are variable upon the region. Rifampicin inhibits bacterial DNA synthesis through the inhibition of the DNA-dependent RNA polymerase
[40]. However, some
M. ulcerans variants previously showed resistance to rifampicin after monotherapy, suggesting that this mycobacterium has the ability to develop resistance against antimicrobial drugs commonly used to treat BU
[41]. Whilst rifampicin and rifabutin have been shown to be the most active drugs against
M. marinum variant infections
[42] they are often treated with tritherapies including rifampicin, clarithromycin and ethambutol
[43][44] thus slightly differing from
M. ulcerans variant therapy. Amikacin, ciprofloxacin, kanamycin and lincomycin inhibited the growth of
M. pseudoshottsii variant isolates
[45]. In addition to being resistant to isoniazid, ethambutol and ethionamide, the
M. liflandii variant also exhibits resistance to rifampicin (rifampin) and clarithromycin
[46][47], suggesting that the combination of these later two antibiotics may not be appropriate to treat BU cases caused by
M. liflandii variants. In French Guiana,
M. gilvum infections were cured either after erysipelas treatment (based on penicillin administration) or with a combination of rifampicin (600 mg daily) and clarithromycin (500 mg twice daily)
[17] while this mycobacterium has been shown to be resistant to isoniazid, sodium aminosalicylate and rifampicin
[48]. These findings suggest that common combinations of antibiotic molecules locally used to treat BU might be inappropriately used depending on the variant/species causing disease.