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A strategy based on Amplified Fragment Length Polymorphism (AFLP) for routine genotyping of nontuberculous mycobacteria at the clinical laboratory

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Abstract

The increasing worldwide incidence of mycobacteriosis and the need to achieve improved clinical management makes nontuberculous mycobacteria (NTM) genotyping a useful tool. However, because of technical difficulties, medium size microbiology laboratories do not attempt to compare the genetic patterns that each of their isolates present. We have aimed to optimize a genotyping method with a reduced hands-on experimental time and that requires few technical resources. A strategy based on the Amplified Fragment Length Polymorphism (AFLP) methodology was developed using two rare-cutters enzymes (SacI and BglII). One out of seven primers was sequentially used in each amplification reaction that was analyzed by agarose gel electrophoresis. This approach makes it possible the timely genotyping of a moderate number of strains and its characterization without the need of image analysis software. We have genotyped 28 Mycobacterium intracellulare and 4 M. abscessus. Clinical researchers are encouraged to routinely genotype their NTM isolates.

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References

  1. Rivero-Lezcano OM, González-Cortés C, Mirsaeidi M (2019) The unexplained increase of nontuberculous mycobacteriosis. Int J Mycobacteriol 8(1):1–6

    Article  CAS  Google Scholar 

  2. Forbes BA, Hall GS, Miller MB, Novak SM, Rowlinson MC, Salfinger M, Somoskövi A, Warshauer DM, Wilson ML (2018) Practice guidelines for clinical microbiology laboratories: mycobacteria. Clin Microbiol Rev 31(2):e00038–e117

    Article  CAS  Google Scholar 

  3. Phillips MS, von Reyn CF (2001) Nosocomial infections due to nontuberculous mycobacteria. Clin Infect Dis 33(8):1363–1374

    Article  CAS  Google Scholar 

  4. Iketleng T, Lessells R, Dlamini MT, Mogashoa T, Mupfumi L, Moyo S, Gaseitsiwe S, de Oliveira T (2018) Mycobacterium tuberculosis next-generation whole genome sequencing: opportunities and challenges. Tuberc Res Treat 2018:1298542

    PubMed  PubMed Central  Google Scholar 

  5. Jagielski T, van Ingen J, Rastogi N, Dziadek J, Mazur PK, Bielecki J (2014) Current methods in the molecular typing of Mycobacterium tuberculosis and other mycobacteria. Biomed Res Int 2014:645802

    PubMed  PubMed Central  Google Scholar 

  6. Jagielski T, Minias A, van Ingen J, Rastogi N, Brzostek A, Żaczek A, Dziadek J (2016) Methodological and clinical aspects of the molecular epidemiology of Mycobacterium tuberculosis and other mycobacteria. Clin Microbiol Rev 29(2):239–290

    Article  CAS  Google Scholar 

  7. Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res 23(21):4407–4414

    Article  CAS  Google Scholar 

  8. Restrepo CM, Llanes A, Lleonart R (2018) Use of AFLP for the study of eukaryotic pathogens affecting humans. Infect Genet Evol 63:360–369

    Article  CAS  Google Scholar 

  9. Gaafar A, Unzaga MJ, Cisterna R, Clavo FE, Urra E, Ayarza R, Martín G (2003) Evaluation of a modified single-enzyme amplified-fragment length polymorphism technique for fingerprinting and differentiating of Mycobacterium kansasii type I isolates. J Clin Microbiol 41(8):3846–3850

    Article  CAS  Google Scholar 

  10. Janssen P, Coopman R, Huys G, Swings J, Bleeker M, Vos P, Zabeau M, Kersters K (1996) Evaluation of the DNA fingerprinting method AFLP as an new tool in bacterial taxonomy. Microbiology 142(Pt 7):1881–1893

    Article  CAS  Google Scholar 

  11. Telenti A, Marchesi F, Balz M, Bally F, Böttger EC, Bodmer T (1993) Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J Clin Microbiol 31(2):175–178

    Article  CAS  Google Scholar 

  12. Dauchy FA, Dégrange S, Charron A, Dupon M, Xin Y, Bébéar C, Maugein J (2010) Variable-number tandem-repeat markers for typing Mycobacterium intracellulare strains isolated in humans. BMC Microbiol 10:93

    Article  Google Scholar 

  13. Hunter PR, Gaston MA (1988) Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. J Clin Microbiol 26(11):2465–2466

    Article  CAS  Google Scholar 

  14. Mokrousov I (2017) Revisiting the Hunter Gaston discriminatory index: note of caution and courses of change. Tuberculosis (Edinb) 104:20–23

    Article  Google Scholar 

  15. Wayne LG, Sramek HA (1992) Agents of newly recognized or infrequently encountered mycobacterial diseases. Clin Microbiol Rev 5(1):1–25

    Article  CAS  Google Scholar 

  16. Viader-Salvadó JM, Flores-Gracia J, Vega-Alonso AS, Treviño-Alvarado VM, Molina-Torres CA, Vera-Cabrera L, Guerrero-Olazarán M (2009) Simplified amplified-fragment length polymorphism method for genotyping Mycobacterium tuberculosis isolates. J Microbiol Methods 78(3):331–338

    Article  Google Scholar 

  17. Jiménez Arias AP, Lahiguera MJ, Borrás R, Gimeno Cardona C, Grijalva Silva M, Vallejo López MJ, Guna Serrano MR (2018) Comparative study of the genetic diversity of Mycobacterium tuberculosis complex by simplified amplified fragment length polymorphism and mycobacterial interspersed repetitive unit variable number tandem repeat analysis. Rev Ecuat Med Cienc Biol 39(1):63–71

    Google Scholar 

  18. Roetzer A, Diel R, Kohl TA, Rückert C, Nübel U, Blom J, Wirth T, Jaenicke S, Schuback S, Rüsch-Gerdes S, Supply P, Kalinowski J, Niemann S (2013) Whole genome sequencing versus traditional genotyping for investigation of a Mycobacterium tuberculosis outbreak: a longitudinal molecular epidemiological study. PLoS Med 10(2):e1001387

    Article  Google Scholar 

  19. Trovato A, Baldan R, Costa D, Simonetti TM, Cirillo DM, Tortoli E (2017) Molecular typing of Mycobacterium Abscessus isolated from cystic fibrosis patients. Int J Mycobacteriol 6(2):138–141

    Article  CAS  Google Scholar 

  20. Jeon S, Lim N, Kwon S, Shim T, Park M, Kim BJ, Kim S (2014) Molecular typing of Mycobacterium intracellulare using pulsed-field gel electrophoresis, variable-number tandem-repeat analysis, mycobacteria interspersed repetitive-unit-variable-number tandem repeat typing, and multilocus sequence typing: molecular characterization and comparison of each typing methods. Osong Public Health Res Perspect 5(3):119–130

    Article  Google Scholar 

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Acknowledgements

This work was supported by Consejería de Sanidad de la Junta de Castilla y León GRS 1225/A/16. Gonzalez‑Cortés was supported by a grant from Ministerio de Economía y Competitividad, subprogram of technical support staff, 2015 (PTA2015‑11248‑I).

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Authors and Affiliations

  1. Servicio de Análisis Clínicos, Hospital de Palamós, Edificio Fleming, Carrer Hospital 36, 17230, Girona, Spain

    Sara Blanco-Conde

  2. Unidad de Investigación, Hospital Universitario de León, Altos de Nava, s/n, 24080, León, Spain

    Carolina González-Cortés & Octavio Miguel Rivero-Lezcano

  3. Servicio de Microbiología Clínica, Hospital Universitario de León, Altos de Nava, s/n, 24080, León, Spain

    Ramiro López-Medrano

  4. Unidad de Referencia Regional de Micobacterias, Servicio de Microbiología, Hospital Universitario Central de Asturias, Avda. Roma s/n, 33011, Oviedo, Asturias, Spain

    Juan José Palacios-Gutiérrez

  5. Servicio de Anatomía Patológica, Hospital Universitario de León, Altos de Nava, s/n, 24080, León, Spain

    Cristina Diez-Tascón

  6. Servicio de Microbiología, Hospital Clínico Universitario de Valladolid, C/ Ramon y Cajal 3, 47005, Valladolid, Spain

    Teresa Nebreda-Mayoral

  7. Servicio de Análisis Clínicos, Hospital Universitario de León, Altos de Nava, s/n, 24080, León, Spain

    María Josefa Sierra-García

  8. Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain

    Octavio Miguel Rivero-Lezcano

  9. Institute of Biomedicine, University of León, León, Spain

    Octavio Miguel Rivero-Lezcano

Authors
  1. Sara Blanco-Conde
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  2. Carolina González-Cortés
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  3. Ramiro López-Medrano
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  4. Juan José Palacios-Gutiérrez
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  5. Cristina Diez-Tascón
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  6. Teresa Nebreda-Mayoral
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  7. María Josefa Sierra-García
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  8. Octavio Miguel Rivero-Lezcano
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Corresponding author

Correspondence to Octavio Miguel Rivero-Lezcano.

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All authors declare that they have not conflict of interest.

Ethical approval

This research was approved by the Comité de Ética de Investigación con medicamentos (CEIm) del Área de Salud de León y El Bierzo (Spain). Reference number: 1784. It does not contain any studies conducted on human or animal subjects.

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Blanco-Conde, S., González-Cortés, C., López-Medrano, R. et al. A strategy based on Amplified Fragment Length Polymorphism (AFLP) for routine genotyping of nontuberculous mycobacteria at the clinical laboratory. Mol Biol Rep 47, 3397–3405 (2020). https://doi.org/10.1007/s11033-020-05420-8

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  • DOI: https://doi.org/10.1007/s11033-020-05420-8

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