A protocol for culturing environmental strains of the Buruli ulcer agent, Mycobacterium ulcerans

Dezemon Zingue,Michel Drancourt,Arup Panda

doi:10.1038/s41598-018-25278-y

Abstract

Contaminations and fastidiousness of M. ulcerans may have both hamper isolation of strains from environmental sources. We aimed to optimize decontamination and culture of environmental samples to circumvent both limitations. Three strains of M. ulcerans cultured onto Middlebrook 7H10 at 30 °C for 20 days yielded a significantly higher number of colonies in micro-aerophilic atmosphere compared to ambient atmosphere, 5% CO2 and anaerobic atmosphere. In a second step, we observed that M. ulcerans genome uniquely encoded chitinase, fucosidase and A-D-GlcNAc-diphosphoryl polyprenol A-3-L-rhamnosyl transferase giving M. ulcerans the potential to metabolize chitine, fucose and N-acetyl galactosamine (NAG), respectively. A significant growth-promoting effect of 0.2 mg/mL chitin (p < 0.05), 0.01 mg/mL N-acetyl galactosamine (p < 0.05), 0.01 mg/mL fucose (p < 0.05) was observed with M. ulcerans indicating that NAG alone or combined with fucose and chitin could complement Middlebrook 7H10. Finally, the protocol combining 1% chlorhexidine decontamination with micro-aerophilic incubation on Middlebrook 7H10 medium containing chitin (0.2%), NAG (0.01%) and fucose (0.01%) medium and auto-fluorescence detection of colonies allowed for the isolation of one mycolactone-encoding strain from Thryonomys swinderianus (aulacode) feces specimens collected near the Kossou Dam, Côte d’Ivoire. We propose that incubation of chlorhexidine-decontaminated environmental specimens on Middlebrook 7H10-enriched medium under micro-aerophilic atmosphere at 30 °C may be used for the tentative isolation of M. ulcerans strains from potential environmental sources.

Highlights

Buruli ulcer is a World Health Organization (WHO)-notifiable, yet neglected infection of the cutaneous and subcutaneous tissues caused by the nontuberculous Mycobacterium ulcerans[1]
Only one environmental M. ulcerans (M. ulcerans 00–1441 from a Buruli ulcer endemic area in Benin, West Africa) isolate has been firmly confirmed on Löwenstein-Jensen medium after 15-day of incubation in BACTEC 12b broth and three successive passages in mouse footpad P1, P2 and P3 for nine months, six months and 12 months, followed by culture on Löwenstein-Jensen for two months[21]
The fact that only a few environmental isolates have been made after such a long experiment, suggests that contamination by fast-growing bacteria or mycobacteria and fungi of the environmental samples along with poorly appropriate culture media limited the isolation of M. ulcerans from environmental sources

Summary

Introduction

Buruli ulcer is a World Health Organization (WHO)-notifiable, yet neglected infection of the cutaneous and subcutaneous tissues caused by the nontuberculous Mycobacterium ulcerans[1]. Three additional reported strains included two IS2404-PCR positive strains from two samples of aquatic plants and two wild aquatic insects collected in a Buruli ulcer endemic area of Côte d’Ivoire[31,32] and two M. ulcerans strains from moss and soil in Ghana[33]. None of these strains have been deposited in public collection. In the perspective of achieving a culture-based field investigation of M. ulcerans, we aimed at improving the decontamination of samples along with the composition of culture media in order to optimize the chance of recovering additional environmental M. ulcerans isolates

Objectives

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Conclusion