Phenotypic and Genotypic Characterization of Mycobacteria Isolates from Buruli Ulcer Suspected Patients Reveals the Involvement of Several Mycobacteria in Chronic Skin Lesions

Abstract

Buruli ulcer is a cutaneous mycobacterial disease that occurs in tropical countries in sub-Saharan Africa, South-East Asia, Australia and America. The responsible pathogen is Mycobacterium ulcerans. Cote d'Ivoire is the most affected country, with more than 30 endemic health districts reporting a large number of chronic skin lesions. The clinical forms and the severity of ulcers vary from one patient to another. Samples from suspected patients were analyzed by PCR at Pasteur Institute of Cote d'Ivoire, as recommended by WHO. IS2404 sequence was detected in 61% of cases, incriminating M. ulcerans in chronic cutaneous lesions. For the other cases the etiology was not identified, thus raising several questions. Are all reported "Buruli ulcer" cases really caused by M. ulcerans? Would other mycobacteria be involved in the occurrence of chronic skin lesions considered as "Buruli ulcer"? BU suspected patients were enrolled in endemic areas of Cote d’Ivoire. Samples were collected from cutaneous lesions and transported to the lab at +4°C in 2 ml of Middlebrook 7H9 medium supplemented by Cetylpiridium chloride. The centrifugation pellet was taken with saline buffer to perform microscopic examination and mycobacteria isolation on Lowenstein-Jensen medium. Biochemical characteristics were described by the nicotinic acid detection according to Konno protocol, by the Nitrates reduction test, by the catalase activity detection at 22° and 68°C and the Wayne's Tween 80 hydrolysis test. Genotypic characteristics were determined by PCR with 1 ml of bacterial suspension targeting the insertion sequences (IS6110, IS2404, and IS2606), the plasmid virulence genes and Miru-VNTR loci (Miru-1, VNTR 6, VNTR 19, ST-1). A total of 47 mycobacterial strains were isolated with 3 different types of colonies whose microscopic examination showed Acid-Alcohol-Resistant Bacilli. 65.9% of isolates expressed biochemical characters in favor of M. ulcerans strains and 6.4% in favor of M. marinum strains. For 29.8% of isolates, the characteristics were related to atypical mycobacterial species. The genotyping targeting the IS6110, IS2606 and IS2404 insertion sequences allowed simultaneous amplification in 53.2% of isolates. IS2404 was amplified in 93.6% of isolates; IS6110 was amplified in 74.5% of isolates and IS2606 was amplified in 70.2% of isolates. Five genotypes were identified corresponding to various species of mycobacteria: genotypes 1 and 2 accounted for 63.8% with all the 3 insertion sequences and biochemical characteristics in favor of M. ulcerans strains; genotype 4 accounted for 6.4% of isolates with insertion sequences and biochemical characteristics in favor of M. marinum strains; The strains of genotypes 3 and 5 expressed molecular and biochemical characters relating to various non-M. ulcerans mycobacteria. Virulence genes were found in 72.3% of isolates corresponding to 90% of M. ulcerans strains and 60.7% of non-M. ulcerans mycobacteria. This study confirmed the involvement of several genotypes of M. ulcerans and other mycobacteria in chronic cutaneous lesions suspected as cases of Buruli ulcer in Cote d’Ivoire.