Non-tuberculous mycobacteria isolates from patients with chronic pulmonary disease and no epidemiological relationship show sequence clusters by whole genome sequencing

Marc Rubio ^1,2 Mariana Fernandez-Pittol ^3,4,5 Sara Batista ^3,5,6 Diego Martínez ⁵ Lorena San Nicolas ⁵ Elena Portell-Buj ^3,4,5 M Antonia Busquets ^7,8 Joan Estelrich ^7,8 Julian Gonzalez-Martin ^3,4,5,9 Ferran Navarro ^1,10,2 Griselda Tudó ^3,4,5 Montserrat Garrigó ^1,10,2*

• ¹ Sant Pau Institute for Biomedical Research, Barcelona, Catalonia, Spain
• ² Servei de Microbiologia, Fundació de Gestió de l'Hospital de la Santa Creu i Sant Pau, Barcelona, Balearic Islands, Spain
• ³ Departament de Fonaments Clinics, Faultat de Medicina i Ciencies de la Salut, Universitat de Barcelona, Barcelona, Spain
• ⁴ Instituto Salud Global Barcelona (ISGlobal), Barcelona, Catalonia, Spain
• ⁵ Servei de Microbiologia, Hospital Clinic de Barcelona, Barcelona, Spain
• ⁶ August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Barcelona, Catalonia, Spain
• ⁷ Departament de Farmàcia, Tecnologia Farmacèutica i Físicoquímica, Facultat de Farmàcia i Ciències de l'Alimentació, Universitat de Barcelona, Barcelona, Spain
• ⁸ Institut de Nanociència i Nanotecnologia, IN2UB, Facultat de Química, Universitat de Barcelona, Barcelona, Spain
• ⁹ CIBER of Infectiuos Diseases (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain
• ¹⁰ Department of Genetics and Microbiology, Faculty of Biosciences, Autonomous University of Barcelona, Barcelona, Catalonia, Spain

To study the genomic epidemiology of slow growing mycobacteria (SGM) isolates from patients with bronchiectasis by whole genome sequencing (WGS) and evaluate different bioinformatic tools to establish relationships between isolates.Forty-six SGM isolates from 37 patients with underlying chronic pulmonary disease, previously identified as Mycobcterium avium, M. intracellulare or M. chimaera by polymerase chain reaction were analysed using WGS and three different clustering methods: rPinecone, split k-mer analysis and custom single nucleotide variant threshold calculation.The three analyses showed one cluster of M. intracellulare subsp. intracellulare isolates and one cluster of M. intracellulare subsp. chimaera isolates from different patients. The analyses did not show any clusters formed by M. avium subsp. avium isolates from different patients.M. intracellulare subsp. chimaera and M. intracellulare subsp. intracellulare form clusters of very closely related isolates from patients with no epidemiologic relationship. This lack of epidemiological relationship suggested acquisition from common sources, rather than direct transmission between patients. The use of the three methodologies of analysis are an adequate strategy to study in depth the relationship between isolates belonging to very close species and subspecies.