Abstract:
Buruli ulcer (BU) is a skin disease caused by Mycobacterium ulcerans. BU is the third most common mycobacterial disease after tuberculosis and leprosy, but in Ghana and Cote d’ Ivoire, it is the second. M. ulcerans produces mycolactone, an immunosuppressant macrolide toxin which makes the infection painless. However, some patients have complained of painful lesions and delay healing. Painful ulcers and delay healing experienced by some patients may be due to secondary bacterial infections. Main Objective: To identify secondary microbial infections of BU patients, their genetic diversity as well as determine the levels of antibiotics resistance of these microorganisms. Methodology: The study was conducted at Biochemistry, Cell and Molecular Biology, University of Ghana. Subjects were recruited from Amansie Central District, Ashanti Region. Swabs of 51 BU patients were taken and immediately frozen for transport into the laboratory. Microscopy was performed using Ziehl-Neelsen and Gram staining techniques. The samples were also cultured on Luria Bertani, MacConkey, Mannitol, BPA and Sabouraud dextrose agar to identify bacteria and fungi. Antibiotic susceptibility tests were performed on selected bacteria species. DNA was extracted from the samples, after which Polymerase Chain Reaction (PCR) was performed using universal (16S rRNA), MSHA/PA (16S rRNA for mycobacterial) and IS2404 (insertion sequence specific to mycolactone producers) primers to find the different strains of organisms. Finally sequencing was performed on the DNA amplicons that were randomly selected to identify the kinds of microorganisms causing secondary infection.
Results: All the samples were positive for bacteria. However 49 and 40 positives were obtained from PCR products using the primers MSHA/PA, and IS2404 respectively, thus 40 BU patients were identified out of the total 51 patient samples. Majority of the bacteria identified after sequencing with universal primers for bacteria were Staphylococcus spp (aureus including MRSA, saprophyticus, and lentus), Alcaligene spp (aquatilis and faecalis), Pseudomonas spp (aeruginosa, stutzeri and koreensis) and bacilli cereus group of bacteria. Interestingly, 60% of the sequencing result for mycobacteria detected the presence of Corynebacterium spp (aurimucosum, diphtheria and striatum). Other bacteria identified were Brevibacterium iodinum and Rhodococcus erythropolis. Majority of these bacteria live in muddy areas and dirty water. The selected bacteria were less susceptible to rifampicin, clarithromycin and amikacin. Conclusion: Other bacteria beside M. ulcerans colonize and proliferate on BU lesions. The selected bacteria were less susceptible to clarithromycin and amikacin and rifampicin. The pains and healing delay experienced by some BU patients could be the result of these bacteria colonizing and proliferating on the ulcer or lesions.
