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

Aka Nguetta^1, , Coulibaly N D¹, Kouamé-Elogne N C¹, Acquah K J R¹, Amon Aby Christiane¹, Kouadio Kouamé¹, Konan N’Guessan², Koffi Aboa³, Kadio M.C³, Yao Aubin², Paul Saunderson⁴, Kakou-Ngazoa ES¹, Faye -Ketté¹ and H et Dosso M¹

¹Department of Research, Pasteur Institute, Abidjan, Côte d’Ivoire

²MAP International, National Office, Abidjan, Côte d’Ivoire

³Department of Health, Buruli Ulcer National Program, Abidjan, Côte d’Ivoire

⁴Head Office, American Leprosy Missions, Greenville, USA

Cite this paper:
Aka Nguetta, Coulibaly N D, Kouamé-Elogne N C, Acquah K J R, Amon Aby Christiane, Kouadio Kouamé, Konan N’Guessan, Koffi Aboa, Kadio M.C, Yao Aubin, Paul Saunderson, Kakou-Ngazoa ES, Faye -Ketté and H et Dosso M. Phenotypic and Genotypic Characterization of Mycobacteria Isolates from Buruli Ulcer Suspected Patients Reveals the Involvement of Several Mycobacteria in Chronic Skin Lesions. American Journal of Microbiological Research. 2018; 6(3):79-87. doi: 10.12691/ajmr-6-3-3

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. Côte 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 Côte 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 Côte 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 Côte d’Ivoire.

Keywords:
Buruli ulcer M. ulcerans insertion sequences other mycobacteria virulence genes

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References:

[1]	Grosset J., Boivert H., Truffot-Pernot C. (1989). Mycobactéries. In: Le Minor L, Véron M., ed. Bactériologie médicale. Paris: Flammarion; 1989: 965-1017.
[2]	Marston B J, Diallo M O, Horsburgh C R Jr, Diomande I, Saki M Z, Kanga J M, Patrice G, Lipman H B, Ostroff S M, Good R C (1995) Emergence of Buruli ulcer disease in the Daloa region of Cote d'Ivoire. Am J Trop Med Hyg.; 52; 3: 219-224.
[3]	Meyers W M, Connor D H, McCullough B, Bourland J, Moris R, Proos L (1974) Distribution of Mycobacterium ulcerans infections in Zaire, including the report of new foci. Ann Soc Belg Med Trop; 54: 147-157.
[4]	OMS (2008). Ulcère de Buruli: rapport 2004-2008. Weekly epidemiological record, N°17, 25 April 2008.
[5]	Perraudin M. L., Herrault A., Desbois J. C. (1980) Ulcère cutané à Mycobacterium ulcerans (ulcère de Buruli). Ann Péd; 27: 687-692.
[6]	Faber W R, Bouda-Arias Pereira L M, Zeegelaar J.E, Kolk A H J, Fonteyne P- A, Toonstra J, Portaels F (2000). First reported case of Mycobacterium ulcerans infection in a patient from China. Trans Roy Soc Trop Med Hyg; 94: 277-279.
[7]	Nakanaga K, Hoshino Y, Yotsu RR, Makino M, Ishii N (2011): Nineteen cases of Buruli ulcer diagnosed in Japan from 1980 to 2010. J Clin Microbiol; 49(11): 3829-36.
[8]	Nakanaga K, Ishii N, Suzuki K, Tanigawa K, Goto M et al. (2007) “Mycobacterium ulcerans subsp. shinshuense” isolated from a skin ulcer lesion: identification based on 16S rRNA gene sequencing. J Clin Microbiol; 45; 11: 3840-3843.
[9]	Yotsu RR, Nakanaga K, Hoshino Y, Suzuki K, Ishii N (2012). Buruli ulcer and current situation in Japan: a new emerging cutaneous Mycobacterium infection. J Dermatol; 39(7): 587-93.
[10]	Anne-Caroline C, Tresse C, Casarottoa V, Nicolas B (2013). History, biology and chemistry of Mycobacterium ulcerans infections (Buruli ulcer disease). Nat. Prod. Rep. 30:1527-1567.
[11]	Johnson P D, Azuolas J, Lavender C J, Wishart E, Stinear T P, Hayman J A, Brown L, Jenkin G A, Fyfe J A (2007). Mycobacterium ulcerans in mosquitoes captured during outbreak of Buruli ulcer, southeastern Australia. Emerg Infect Dis; 13; 11: 1653-1660.
[12]	Mac C, Toh JC, Buckle G, Sis-Sons HA (1948). A new bacterial infection in man: clinical aspects. J. Pathol. Bacteriol. 60: 93-101.
[13]	Dos Santos V M, Noronha F L, Vicentina E C, Lima C C (2007) Mycobacterium ulcerans infection in Brazil. Med J Aust; 187: 63-64.
[14]	Guerra H, Palomino J C, Falconí E, Bravo F, Donaires N, Van Marck E, Portaels F (2008) Mycobacterium ulcerans Disease, Peru. Emerg Inf Dis; 14; 3: 373-377.
[15]	McGann H, Stragier P, Portaels F, Gascoyne-Binzi D, Collyns T, Lucas S, Mawer D (2009) Buruli Ulcer in United Kingdom Tourist Returning from Latin America. Emerg Inf Dis; 15; 11: 1847-1859.
[16]	OMS (2013). Recommandations pour la lutte contre l’ulcère de Buruli. Genève 25-27 mars 2013.
[17]	Laurence A, Aka N, Euloge E, Samir B, Raymond N, Mireille D (2009). Risk factors for Buruli ulcer in Côte d’Ivoire: Results of a case-control study. African Journal of Biotechnology Vol. 8 (4), pp. 536-546, 18.
[18]	Eddyani M, Fraga AG, Schmitt F, Uwizeye C, Fissette K, Johnson C, Aguiar J, Sopoh G, Barogui Y, Meyers WM, Pedrosa J, Portaels F (2009). Fine-needle aspiration, an efficient sampling technique for bacteriological diagnosis of non-ulcerative Buruli ulcer. J. Clin. Microbiol. 47(6): 1700-1704.
[19]	Aka N’guetta, KAKOU-NGAZOA E. Solange, COULIBALY N. D., VAKOU Sabine, KOUADIO Kouamé, AOUSSI Serge, KADIO M. Constance, KOUAKOU Hélène, AKE Julien, N’GUESSAN Raymond and DOSSO Mireille (2015). “Virulence factors expressed by Mycobacterium ulcerans strains: Results of a descriptive study”. African Journal of Microbiology Research, Vol. 9(28), pp. 1745-1751.
[20]	Adalbert L, Mohammed A, Gisela B, Fadila B, David D, Lanfranco F, Knut F, Thomas F(2000). (Union) : Diagnostic de la tuberculose par examen microscopique direct des expectorations dans les pays à faibles revenus : Guide technique. Cinquième édition 2000, 60 pages.
[21]	Guide de Bonne Exécution des Examens de Laboratoire (GBEA), N°287, du 11 décembre 1999, page 18441.
[22]	Wilton S, Cousins D (1992). Detection and identification of multiple mycobacterial pathogens by DNA amplification in a single tube. PCR Methods Appl; 1: 269-273.
[23]	Stinear T P, Ross B, Davis J, Marino L, Robins-Browne R, Oppedinaso F, Sievers A, Johnson P D (1999). Identification and characterization of IS2404 and IS2606: two distinct repeated sequences for detection of Mycobacterium ulcerans by PCR. J Clin Microbiol; 37: 1018-1023.
[24]	Anthony Ablordey, Jean Swings, Christine Hubans, Karim Chemlal, Camille Locht, Françoise Portaels and Philip Supply (2005). Multilocus Variable-Number Tandem Repeat Typing of Mycobacterium ulcerans. Journal of clinical Microbiology, p. 1546-1551.
[25]	Stragier P, Ablordey A, Durnez L, Portaels F (2007) VNTR analysis differentiates Mycobacterium ulcerans and IS2404 positive mycobacteria. Syst Appl Microbiol; 30: 525-530.
[26]	Stragier P, Ablordey A, Bayonne L M, Lugor Y L, Sindani I S, Suykerbuyk P, Wabinga H, Meyers W M, Portaels F (2006) Heterogeneity among Mycobacterium ulcerans isolates from Africa. Emerg Infect Dis; 12: 844-847.
[27]	Stragier P, Ablordey A, Meyers W M, Portaels F (2005) Genotyping Mycobacterium ulcerans and Mycobacterium marinum by using mycobacterial interspersed repetitive units. J. Bacteriol; 187: 1639-1647.
[28]	Ehuié Pierre (2000). Diagnostic de l'ulcère de Buruli par la détection de l'IS2606: Une étude expérimentale à partir d'exsudats cutanés provenant de malades résidant dans la région des Lacs en 1998 en Côte d'Ivoire. Mémoire pour le Diplôme d'Ingénieurs des Techniques Sanitaires - Option Microbiologie, 95 p.
[29]	Elise SN, Euloge E, Nguetta A, David CN, Bakary C, Mireille D (2011). Evaluation of real-time PCR for Mycobacterium ulcerans in endemic region in Côte d’Ivoire. African Journal of Microbiology Research. 5(16):2211-2216.
[30]	Fyfe JA, Lavender CJ, Johnson PD, Globan M, Sievers A, Azuolas J, Stinear TP (2007). Development and application of two multiplex real-time PCR assays for the detection of Mycobacterium ulcerans in clinical and environmental samples. Appl. Environ. Microbiol. 73(15): 4733-4740.
[31]	Rondini S, Mensah-Quainoo E, Troll H., Bodmer T, Pluschke, G. (2003). Development and application of real-time PCR assay for quantification of Mycobacterium ulcerans DNA. J Clin Microbiol; 41: 4231-4237.
[32]	Ross B C, Johnson P D, Oppedisano F, Marino L, Sievers A, Stinear T, Hayman J A, Veitch M G, Robins-Browne R M (1997) Mycobacterium ulcerans in environmental samples during an outbreak of ulcerative diseases. Appl Environ Microbiol, 63: 4135-4138.
[33]	Fred S Sarfo, Richard O Phillips, Jihui Zhang, Mohammed K Abass, Justice Abotsi, Yaw A Amoako, Yaw Adu-Sarkodie, Clive Robinson, Mark H Wansbrough-Jones (2014): Kinetics of mycolactone in human subcutaneous tissue during antibiotic therapy for Mycobacterium ulcerans disease. BMC Infect. Dis. 14: 202.
[34]	Fred SS, Fabien LC, N'Guetta A, Richard OP, Yaw A, Ivo GB, Pascal L, Mireille D, Mark WJ, Romain VC, Laure GM, Caroline D (2011). Mycolactone Diffuses into the Peripheral Blood of Buruli Ulcer Patients - Implications for Diagnosis and Disease Monitoring. PLoS Negl. Trop. Dis. 5(7): e1237.
[35]	Quinet G, Kakou-Ngazoa E S, Aka N, Vakou S, Coulibaly N D, Kouakou H, Sylla A, Faye-Kette H, Aoussi S and Dosso M (2017). Molecular Typing using PCR-RFLP Reveals Diversity of Environmental Mycobacteria Agent of Buruli Ulcer in Ivory Coast, Cote d’Ivoire (West Africa). Intern Med, an open access journal, Vol 7: 3.
[36]	Kakou Ngazoa E. Solange, Coulibaly N. David, Aka Nguetta, Vakou Sabine, Aoussi Serge and Dosso Mireille (2015). Clonality of Mycobacterium ulcerans by Using VNTR-MIRU Typing in Ivory Coast (Côte d’Ivoire), West Africa. International Journal of Tropical Disease & Health 7(4): 163-171.
[37]	Stinear T P, Mve-Obiang A, Small P L, Frigui W, Pryor M J, Brosch R, Jenkin G A, Johnson P D R, Davies J K, Lee R E, Adusumilli S, Garnier T, Haydock S F, Leadlay P F, Cole S T ( 2004) Giant plasmid-encoded polyketide synthases produce the macrolide toxin of Mycobacterium ulcerans. Proc Natl Acad Sci U S A, 101; 5: 1345-1349.
[38]	Timothy PS, Melinda JP, Jessica LP, Stewart TC (2005). Functional analysis and annotation of the virulence plasmid pMUM001 from Mycobacterium ulcerans. Microbiology. 151: 683-692.
[39]	Sabine N. Vakou, Julien Coulibaly-Kalpy, Solange E. Kakou-Ngazoa, N`guetta Aka,N`golo D. Coulibaly, Mireille Dosso and Allico J. Djaman (2016). Microbiological and molecular characterization of environmental mycobacterium strains isolated from the Buruli ulcer endemic and non-endemic zones in Cote d’Ivoire. African Journal of Microbiology Research, Vol.10 (40), pp. 1703-1710.
[40]	Oniangue NC (2000) Validation de l'amplification de la séquence d'insertion IS2404 pour le diagnostic de l'ulcère de Buruli en Côte d'Ivoire. Mémoire DEA de Biologie Humaine Tropicale. Option microbiologie. 2000, 66 pages.