Molecular typing of Mycobacterium kansasii using pulsed-field gel electrophoresis and a newly designed variable-number tandem repeat analysis

Paweł Parniewski,Izabela Szulc-Kiełbik,Paulina Borówka,Zofia Bakuła,Anna Żaczek,Jarosław Dziadek,Tomasz Jagielski,Małgorzata Proboszcz,Jakko Van Ingen,Dominik Strapagiel,Agata Podpora,Anna Brzostek,Jacek Bielecki

doi:10.1038/s41598-018-21562-z

Abstract

Molecular epidemiological studies of Mycobacterium kansasii are hampered by the lack of highly-discriminatory genotyping modalities. The purpose of this study was to design a new, high-resolution fingerprinting method for M. kansasii. Complete genome sequence of the M. kansasii ATCC 12478 reference strain was searched for satellite-like repetitive DNA elements comprising tandem repeats. A total of 24 variable-number tandem repeat (VNTR) loci were identified with potential discriminatory capacity. Of these, 17 were used to study polymorphism among 67 M. kansasii strains representing six subtypes (I-VI). The results of VNTR typing were compared with those of pulsed-field gel electrophoresis (PFGE) with AsnI digestion. Six VNTRs i.e. (VNTR 1, 2, 8, 14, 20 and 23) allow to differentiate analyzed strains with the same discriminatory capacities as use of a 17-loci panel. VNTR typing and PFGE in conjunction revealed 45 distinct patterns, including 11 clusters with 33 isolates and 34 unique patterns. The Hunter-Gaston’s discriminatory index was 0.95 and 0.66 for PFGE and VNTR typing respectively, and 0.97 for the two methods combined. In conclusion, this study delivers a new typing scheme, based on VNTR polymorphism, and recommends it as a first-line test prior to PFGE analysis in a two-step typing strategy for M. kansasii.

Highlights

Molecular typing of Mycobacterium kansasii using pulsed-field gel electrophoresis and a newly designed variable-number tandem repeat analysis
Among 1,447 variable-number tandem repeat (VNTR) found in the genome of the M. kansasii American Type Culture Collection (ATCC) 12478 reference strain
In the epidemiology of infectious diseases, including those of mycobacterial etiology, the key issue is disclosing sources of infection, transmission links, and dissemination in the environment. For this to be accomplished, high resolution inter-strain discrimination or typing is of utmost importance[33]

Summary

Introduction

Molecular typing of Mycobacterium kansasii using pulsed-field gel electrophoresis and a newly designed variable-number tandem repeat analysis. The purpose of this study was to design a new, high-resolution fingerprinting method for M. kansasii. A total of 24 variable-number tandem repeat (VNTR) loci were identified with potential discriminatory capacity. 17 were used to study polymorphism among 67 M. kansasii strains representing six subtypes (I-VI). The results of VNTR typing were compared with those of pulsed-field gel electrophoresis (PFGE) with AsnI digestion. The Hunter-Gaston’s discriminatory index was 0.95 and 0.66 for PFGE and VNTR typing respectively, and 0.97 for the two methods combined. This study delivers a new typing scheme, based on VNTR polymorphism, and recommends it as a first-line test prior to PFGE analysis in a two-step typing strategy for M. kansasii. Since only two cases of M. kansasii human-to-human transmission have been documented[1,2], the presumed source of the disease is exposure to environmental reservoirs, municipal tap water[3,4]

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Conclusion