Mycobacterium ulcerans causes Buruli ulcer, an ulcerative skin disease in tropical and subtropical areas. Despite restricted genetic diversity, mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis on M. ulcerans revealed 3 genotypes from different African countries. It is the first time this typing method succeeded directly on patient samples

Ablordey, A

Bayonne, LM

Lugor, YL

Meyers, WM

Portaels, F

Sindani, IS

Stragier, Pieter

Suykerbuyk, P

Wabinga, H

English

Ghent University Academic Bibliography

HeterogeneityamongMycobacteriumulcerans Isolatesfrom Africa Pieter Stragier,* Anthony Ablordey,* L. Manou Bayonne,† Yatta L. Lugor,‡ Ireneaus S. Sindani,§ Patrick Suykerbuyk,*Henry Wabinga,¶ Wayne M. Meyers,# and Françoise Portaels*Mycobacterium ulcerans causes Buruli ulcer, an ulcer-ative skin disease in tropical and subtropical areas. Despiterestricted genetic diversity, mycobacterial interspersedrepetitive unit–variable-number tandem repeat analysis onM. ulcerans revealed 3 genotypes from different Africancountries. It is the first time this typing method succeededdirectly on patient samples. Buruli ulcer (BU), the third most common mycobacter-ial disease after tuberculosis and leprosy, is a majorhealth problem in several West and Central African coun-tries (1). Although endemic in Central America and sub-tropical climates of Southeast Asia and Australia, countriesin Africa in the past decade have recorded increased inci-dence rates in some communities exceeding that of tuber-culosis (2). Mode(s) of transmission, natural reservoir(s) and otherkey aspects of the epidemiology of BU are not fully under-stood, a situation partly complicated by an apparent lack ofgenetic diversity of Mycobacterium ulcerans, as shown byseveral independent genetic markers (3–6). Conventionaland molecular data suggest that M. ulcerans is an environ-mental pathogen because of the selective association ofBU-endemic foci with wetlands and overflowed riverbanks and the detection of M. ulcerans–specific sequencesin water, mud, aquatic insects, and plants (7–9). Specificreservoirs of the etiologic agent cannot be definitivelyassigned; however, we have cultivated M. ulcerans from asingle aquatic insect from Benin (10). Extensive molecular typing of M. ulcerans isolatesrecovered from patients in many endemic foci has beenundertaken to further the understanding of the epidemiolo-gy of BU. A set of robust genotyping methods has alreadybeen applied to M. ulcerans: IS2404 restriction fragmentlength polymorphism (11), amplified fragment lengthpolymorphism analysis (AFLP) (12), multilocus sequencetyping (13), variable-number tandem repeat (VNTR) (3),mycobacterial interspersed repetitive unit (MIRU)–VNTR(6), IS2426 polymerase chain reaction (PCR) (5), andIS2404-Mtb2 PCR (4). All methods, except AFLP, result-ed in geographically related genotypes for China, Japan,Mexico, Suriname, French Guiana, Malaysia, Papua NewGuinea II and Papua New Guinea III, Australia Victoria,Australia Queensland, and Africa. Current typing methodshave established a striking geographic and temporal homo-geneity in African isolates from Angola, Benin,Democratic Republic of Congo (DRC), Ghana, Côted’Ivoire, and Togo (3–6). Even M. ulcerans cultured fromthe insect collected in Benin showed an identical Africangenotype (6). Recently, however, Hilty et al., using aVNTR typing method and sequence analysis, described 3genotypes in Ghana (14). The development of more dis-criminating typing methods may unravel the source andmode of transmission of M. ulcerans and other epidemio-logic aspects of BU.Improved understanding of the molecular biology of M.ulcerans will likely help elucidate observed differences inclinical manifestations. Reported disease recurrence ratesvary from 6% to >20% (15). To what degree this recur-rence is attributable to exogenous reinfection or dissemi-nation of the pathogen from previous lesions is unknown.The relative contribution of variations in pathogen andhost factors to progression and severity of disease likewiseremains obscure.We report the first evidence of genetic diversity in M.ulcerans samples from 3 African countries: DRC, Sudan,and Uganda. Previously, we identified tandem repeat loci,MIRUs (6), and VNTRs (3) in the genome of M. ulcerans.A selection of these MIRUs and VNTRs were used in thisstudy to analyze M. ulcerans extracts from tissue speci-mens from Benin, Togo, Gabon, Uganda, and Sudan, andfrom previous isolates from patients from Cameroon,DRC, Uganda, and Congo-Brazzaville  (Table 1). Resultswere compared with those of a geographically diverse col-lection (n = 39) that were typed in our previous study (6).The StudyTo investigate the MIRU polymorphism, whole genom-ic DNA was prepared from bacterial cultures or clinicalspecimens. The specimens were tissue fragments frompatients with nonulcerated (plaques and edematous forms)or ulcerated forms. DNA extraction from pure cultures wasperformed by heating the colonies in Tris-EDTA at 95°Cfor 10 minutes. Clinical specimens from laboratory-DISPATCHES844 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 5, May 2006*Institute of Tropical Medicine, Antwerp, Belgium; †CentreHospitalier de Libreville, Libreville, Gabon; ‡Yambio Hospital,Eldoret, Kenya; §World Health Organization South Sudan Office,Gigiri, Nairobi, Kenya; ¶Makerere University, Kampala, Uganda;and #Armed Forces Institute of Pathology, Washington, DC, USAconfirmed cases of BU were decontaminated by using thereversed Petroff method, and mycobacterial DNA wasextracted from the decontaminated solution as previouslydescribed (6). Smears of the suspensions were stained bythe Ziehl-Neelsen method.PCR was run as previously described (6). The Agilent2100 Bioanalyzer system (Agilent Technologies,Waldbronn, Germany) was used to separate 1 µL of PCRproduct electrophoretically. Comparison of MIRU-VNTR copy numbers using 4loci showed 11 different profiles. M. ulcerans isolates fromDRC and Uganda and tissue extracts from patients fromSudan (Nzara) and Uganda (Nakasongola) showed distinctprofiles (Central Africa: 1222 and East Africa: 4111), dif-ferent from the originally homogeneous African genotype(Atlantic Africa: 3113; Table 1). In DRC, 3 different geno-types exist, corresponding to 3 different provinces: Bas-Congo, Maniema (Kasongo), and Orientale (Bunia). Theisolate from Orientale was from near the Ugandan border(Lake Albert). Isolates from Gabon, Congo-Brazzaville,and Cameroon had the typical African genotype, now des-ignated the Atlantic African genotype. Identical MIRU-VNTR profiles were observed by using DNA extractedfrom tissues or cultures from patients residing in the samearea. The specificity of the MIRU-VNTR method was test-ed on 14 different Mycobacterium spp. Only M. marinum,M. shottsii, and M. liflandii tested positive, but they weredistinguished from M. ulcerans by exhibiting differentprofiles (data not shown). Sequencing of the concernedloci showed the conserved MIRU sequence at locus 1 and9 in M. ulcerans. Locus 6 (3) and locus 33 contain respec-tively a 56-bp and a 58-bp tandem repeat (Table 2).Heterogeneity among Mycobacterium ulcerans IsolatesEmerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 5, May 2006 845ConclusionsAlthough M. ulcerans isolates from Africa are relative-ly homogeneous, this study demonstrates more hetero-geneity between strains than previously reported. Allisolates from West Africa (Côte d’Ivoire, Ghana, Togo,Benin) and Central Africa (Cameroon; Gabon; Congo-Brazzaville; DRC Bas-Congo; Angola) have the identicalMIRU-VNTR profile, and all originate from regions (i.e.,Bas-Congo) or countries that border the Atlantic Ocean.The isolates that come from regions or countries in theNile River basin (i.e., Orientale in DRC, Sudan, andUganda) or the Congo River basin (i.e., Maniema) havedistinct profiles. These results demonstrate for the first time hetero-geneity among M. ulcerans from different African coun-tries. The 3 African profiles are the Atlantic Africanprofile, the Central African Congo River basin profile, andthe East African Nile River basin profile. This is also thefirst detection of MIRUs and VNTRs in clinical speci-mens, even in smear-negative specimens. These data show that MIRUs and VNTRs are helpfultools in genotyping M. ulcerans. Further detailed differen-tiation of this etiologic agent will lead to an understandingof the epidemiology of BU. As in tuberculosis, better dis-criminatory typing methods help assess the efficacy ofantimycobacterial treatment of BU patients by differentiat-ing reactivation from reinfection. Although M. ulceransappears to be quite monomorphic, full sequencing of thisorganism will permit detection of genes specific for M.ulcerans, and more discriminatory VNTR should becomeavailable.AcknowledgmentsWe thank J. Noeske for providing M. ulcerans isolates fromCameroon and K. Fissette and E. Keijmel for maintaining cul-tures.This work was partly supported by the Directorate-Generalfor the Development Cooperation (Brussels, Belgium) Project:Lutte contre la tuberculose et l’ulcère de Buruli au Bénin); by theDamien Foundation (Brussels, Belgium), and by the EuropeanCommission, project no. INCO-CT-2005-051476-BURULICO“Buruli ulcer: multidisciplinary research for improvement ofcontrol in Africa.” Mr Stragier is a doctoral student at the MycobacteriologyUnit, Institute of Tropical Medicine in Antwerp, Belgium. Hisresearch focuses on molecular microbiology and epidemiologicand environmental aspects of M. ulcerans. He is currentlyengaged in the Burulico project of the European Union toimprove control of BU in Africa.References 1. Portaels F. Epidemiology of mycobacterial diseases. In: Schuster M,editor. Mycobacterial diseases of the skin. Clinics in dermatology.New York: Elsevier Science Inc.; 1995. p. 207–22.2. Debacker M, Aguiar J, Steunou C, Zinsou C, Meyers WM, GuédénonA, et al. Mycobacterium ulcerans disease (Buruli ulcer) in rural hos-pital, southern Benin, 1997–2001. Emerg Infect Dis. 2004;10:1391–8.3. Ablordey A, Swings J, Hubans C, Chemlal K, Locht C, Portaels F, etal. Multilocus variable-number tandem repeat typing ofMycobacterium ulcerans. J Clin Microbiol. 2005;43:1546–51.4. Ablordey A, Kotlowski R, Swings J, Portaels F. PCR amplificationwith primers based on IS2404 and GC-rich repeated sequence revealspolymorphism in Mycobacterium ulcerans. J Clin Microbiol.2005;43:448–51.5. Stinear T, Davies JK, Jenkin GA, Hayman JA, Portaels F, Ross BC,et al. A simple PCR method for rapid genotype analysis ofMycobacterium ulcerans. J Clin Microbiol. 2000;38:1482–7.6. Stragier P, Ablordey A, Meyers WM, Portaels F. GenotypingMycobacterium ulcerans and M. marinum by using mycobacterialinterspersed repetitive units. J Bacteriol. 2005;187:1639–47.7. Marsollier L, Stinear T, Aubry J, André JPS, Robert R, Legras P, et al.Aquatic plants stimulate the growth of and biofilm formation byMycobacterium ulcerans in axenic culture and harbor these bacteriain the environment. Appl Environ Microbiol. 2004;70:1097–103.8. Portaels F, Elsen P, Guimaraes-Peres A, Fonteyne PA, Meyers MW.Insects in the transmission of Mycobacterium ulcerans infection.Lancet. 1999;353:986.9. Portaels F, Chemlal K, Elsen P, Johnson PDR, Hayman JA, KirkwoodR, et al. Mycobacterium ulcerans in wild animals. In: Collins MT,Manning B. Mycobacterial infections in domestic and wild animals.Paris: Office International des Epizooties; 2001. p. 252–64.10. Chemlal K, Huys G, Laval F, Vincent V, Savage C, Gutierrez C, et al.Characterization of an unusual Mycobacterium: a possible missinglink between Mycobacterium marinum and Mycobacterium ulcerans.J Clin Microbiol. 2002;40:2370–80.11. Chemlal K, De Ridder K, Fonteyne PA, Meyers WM, Swings J,Portaels F. The use of IS2404 restriction fragment length polymor-phisms suggests the diversity of Mycobacterium ulcerans from differ-ent geographical areas. Am J Trop Med Hyg. 2001;64:270–3.12. Huys G, Rigouts L, Chemlal K, Portaels F, Swings J. Evaluation ofamplified fragment length polymorphism analysis for inter- andintraspecific differentiation of Mycobacterium bovis, M. tuberculosis,and M. ulcerans. J Clin Microbiol. 2000;38:3675–80.DISPATCHES846 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 5, May 200613. Stinear T, Jenkin GA, Johnson PD, Davies JK. Comparative geneticanalysis of Mycobacterium ulcerans and Mycobacterium marinumreveals evidence of recent divergence. J Bacteriol.2000;182:6322–30.14. Hilty M, Yeboah-Manu D, Boakye D, Mensah-Quainoo E, Rondini S,Schelling E, et al. Genetic diversity in Mycobacterium ulcerans iso-lates from Ghana revealed by a newly identified locus containing avariable number of tandem repeats. J Bacteriol. 2006;188:1462–5.15. Debacker M, Aguiar J, Steunou C, Zinsou C, Meyers WM,  PortaelsF. Buruli ulcer recurrence, Benin. Emerg Infect Dis. 2005;11:584–9.Address for correspondence: Françoise Portaels, Department ofMicrobiology, Mycobacteriology Unit, Institute of Tropical Medicine,Nationalestraat 155, 2000 Antwerpen, Belgium; email: portaels@itg.beHeterogeneity among Mycobacterium ulcerans IsolatesEmerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 5, May 2006 847Searchpast issues

Heterogeneity among Mycobacterium ulcerans isolates from Africa

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Heterogeneity among Mycobacterium ulcerans isolates from Africa

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