Background We describe two contamination cases of Mycobacterium ulcerans clinically infected lesions by Rhodococcus erythropolis, a bacterium of environmental origin with rare cases of human infection.

Methods Two lesion swabs collected from clinically characterized Buruli ulcer-like patients were submitted to molecular (IS2404-PCR) and biological (OA-decontamination + microscopy + culture) analyses for detection and in-vitro culture of Mycobacterium ulcerans (Buruli ulcer etiological agent) respectively.

Results Analysis of DNA extracts from crude samples revealed the presence of M. ulcerans DNA and the sample classified as positive. After 14-days of in-vitro incubation in Löwenstein-Jensen culture media, yellow colonies characteristic of M. ulcerans were observed. However, PCR analysis of culture suspensions revealed no M. ulcerans DNA, while Ziehl-Neelsen staining showed rough red colonies not characteristic of M. ulcerans. Further microbial identification and characterization by MALDI-TOF MS revealed the presence of Rhodococcus erythropolis. The identification was confirmed by whole-genome sequencing (WGS) to establish the genomic link between this originally called Mycobacterium erythropolis and M. ulcerans. Blasting of short reads from WGS confirmed the organism as R. erythropolis. Comparative analysis of whole genome sequences revealed low genomic relatedness between the two organisms with 5.72% average genome coverage using M. ulcerans Agy99 reference genome.

Conclusion This cases illustrates that Buruli ulcer disease may be underdiagnosed due to lesion contamination by R. erythropolis and difficulties in M. ulcerans identification in the routine clinical diagnosis procedure.

Funding: This project was funded by the EDCTP2 programme supported by the EU [TMA2019PF-2693-AGBBU].