Investigation of skin microbiota reveals Mycobacterium ulcerans-Aspergillus sp. trans-kingdom communication.

Mycobacterium ulcerans secrete a series of non-ribosomal-encoded toxins known as mycolactones that are responsible for causing a disabling ulceration of the skin and subcutaneous tissues named Buruli ulcer. The disease is the sole non-contagion among the three most common mycobacterial diseases in humans. Direct contact with contaminated wetlands is a risk factor for Buruli ulcer, responsible for M. ulcerans skin carriage before transcutaneous inoculation with this opportunistic pathogen. In this study, we analysed the bacterial and fungal skin microbiota in individuals exposed to M. ulcerans in Burkina Faso. We showed that M. ulcerans-specific DNA sequences were detected on the unbreached skin of 6/52 (11.5%) asymptomatic farmers living in Sindou versus 0/52 (0%) of those living in the non-endemic region of Tenkodogo. Then, we cultured the skin microbiota of asymptomatic M. ulcerans carriers and negative control individuals, all living in the region of Sindou. A total of 84 different bacterial and fungal species were isolated, 21 from M. ulcerans-negative skin samples, 31 from M. ulcerans-positive samples and 32 from both. More specifically, Actinobacteria, Aspergillus niger and Aspergillus flavus were significantly associated with M. ulcerans skin carriage. We further observed that in vitro, mycolactones induced spore germination of A. flavus, attracting the fungal network. These unprecedented observations suggest that interactions with fungi may modulate the outcome of M. ulcerans skin carriage, opening new venues to the understanding of Buruli ulcer pathology, prophylaxis and treatment of this still neglected tropical infection.

Automatic microscopic detection of mycobacteria in sputum: a proof-of-concept.

The laboratory diagnosis of lung mycobacterioses including tuberculosis comprises the microscopic examination of sputum smear after appropriate staining such as Ziehl-Neelsen staining to observe acid-fast bacilli. This standard procedure is operator-dependant and its sensitivity depends on the duration of observation. We developed and evaluated an operator-independent microscopic examination of sputum smears for the automated detection and enumeration of acid-fast bacilli using a ZEISS Axio Scan.Z1 microscope. The sensitivity, specificity, positive predictive value, negative predictive values and accuracy were calculated using standard formulations by comparison with standard microscopic examination. After in-house parameterization of the automatic microscope and counting software, the limit of detection evaluated by seeding negative sputa with Mycobacterium bovis BCG or Mycobacterium tuberculosis H37Rv (100-105 bacilli/mL) was of 102 bacilli/mL of sputum with a 100% positivity rate. Then, the evaluation of 93 sputum specimens including 34 smear-positive and 59 smear-negative specimens yielded a sensitivity of 97.06% [84.67-99.93%], a specificity of 86.44% [73.01-92.78%]. Up to 100 smear slides could be stocked for reading in the microscope magazine and results are exportable into the laboratory information system. Based on these preliminary results, we are implanting this automatic protocol in the routine workflow so that only smears detected positive by automatic microscopy are confirmed by standard microscopic examination.

Direct matrix-assisted laser desorption ionisation time-of-flight mass spectrometry identification of mycobacteria from colonies.

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) identification of mycobacteria requires a standard acetonitrile/formic acid pre-MALDI-TOF-MS. We prospectively compared this standard protocol with direct deposit with matrix for the identification of mycobacteria cultured on solid media. We first verified that Mycobacterium tuberculosis was killed after it was mixed with matrix. Then, 111 Mycobacterium isolates previously identified by partial rpoB gene sequencing were tested in parallel by the two protocols. An identification score >1.7 was obtained in 86/111 (77.5 %) isolates after protein extraction versus 97/111 (87.4 %) isolates after direct deposit (p = 0.039, Chi-squared test). In a third step, we determined that direct deposit achieved identification for as few as 2.104 M. tuberculosis organisms. In a fourth step, we evaluated direct deposit of one colony for 116 solid medium-cultured clinical isolates finally identified as representative of 12 species (63.8 % M. tuberculosis). For 114/116 (98.3 %) isolates with an identification score >1.2, the MALDI-TOF-MS identification was in complete agreement with the reference rpoB gene sequencing identification. One isolate with a MALDI-TOF-MS identification score of 1.22 for M. fortuitum was identified as M. avium by partial rpoB gene sequencing. One other isolate with a MALDI-TOF-MS identification score of 1.22 for M. tuberculosis was identified as M. tuberculosis by genotyping. All the original MALDI-TOF-MS spectra reported here have been deposited in a public database. Direct deposit of one colony on a MALDI-TOF-MS plate allows for an accurate identification of mycobacteria for an identification score >1.3.