16S-23S Internal Transcribed Spacer Region PCR and Sequencer-Based Capillary Gel Electrophoresis has Potential as an Alternative to High Performance Liquid Chromatography for Identification of Slowly Growing Nontuberculous Mycobacteria.

Shradha Subedi,Sharon C-A Chen,Peter Jelfs,Meng Xiao,Fanrong Kong,Vitali Sintchenko,Timothy J Gray,Luis Angel Maldonado Manjarrez

doi:10.1371/journal.pone.0164138

Abstract

Accurate identification of slowly growing nontuberculous mycobacteria (SG-NTM) of clinical significance remains problematic. This study evaluated a novel method of SG-NTM identification by amplification of the mycobacterial 16S-23S rRNA internal transcribed spacer (ITS) region followed by resolution of amplified fragments by sequencer-based capillary gel electrophoresis (SCGE). Fourteen American Type Culture Collection (ATCC) strains and 103 clinical/environmental isolates (total n = 24 species) of SG-NTM were included. Identification was compared with that achieved by high performance liquid chromatography (HPLC), in-house PCR and 16S/ITS sequencing. Isolates of all species yielded a SCGE profile comprising a single fragment length (or peak) except for M. scrofulaceum (two peaks). SCGE peaks of ATCC strains were distinct except for peak overlap between Mycobacterium kansasii and M. marinum. Of clinical/environmental strains, unique peaks were seen for 7/17 (41%) species (M. haemophilum, M. kubicae, M. lentiflavum, M. terrae, M. kansasii, M. asiaticum and M. triplex); 3/17 (18%) species were identified by HPLC. There were five SCGE fragment length types (I–V) each of M. avium, M. intracellulare and M. gordonae. Overlap of fragment lengths was seen between M. marinum and M. ulcerans; for M. gordonae SCGE type III and M. paragordonae; M. avium SCGE types III and IV, and M. intracellulare SCGE type I; M. chimaera, M. parascrofulaceum and M. intracellulare SCGE types III and IV; M. branderi and M. avium type V; and M. vulneris and M. intracellulare type V. The ITS-SCGE method was able to provide the first line rapid and reproducible species identification/screening of SG-NTM and was more discriminatory than HPLC.

Highlights

Growing nontuberculous mycobacteria (SG-NTM) are environmental bacteria that can cause lung, lymph node, bone, skin and soft tissue, as well as disseminated, infections [1,2,3,4]
The profile of M. avium American Type Culture Collection (ATCC) 25291 when analysed by the two different softwares, and on two independent test occasions yielded a single peak at 368 bp
By studying a large number of Slowly growing nontuberculous mycobacteria (SG-NTM) isolates representing 24 species, we demonstrated that an internal transcribed spacer (ITS)-targeted Sequencer-based capillary gel electrophoresis (SCGE) assay was able to rapidly and reliably identified 13 species and has potential as an alternative to high performance liquid chromatography (HPLC)-based identification of clinically relevant mycobacteria

Summary

Introduction

Growing nontuberculous mycobacteria (SG-NTM) are environmental bacteria that can cause lung, lymph node, bone, skin and soft tissue, as well as disseminated, infections [1,2,3,4]. SG-NTM colonize the airways of patients with chronic lung disease and are increasingly encountered in respiratory tract specimens [1, 6, 7]. The spectrum of recognized SG-NTM species (over 160) is ever widening (http://www.bacterio.net/mycobacterium.html) with evolving taxonomy, re-definition of species complex and identification of individual species within complexes [4, 8, 9]; an increasing number may cause human disease [1]. The most common pathogenic species or species groups include Mycobacterium avium complex (MAC; comprising M. avium, M. intracellulare and other species), M. simiae complex, M. terrae complex, M. haemophilum, M. kansasii and M. marinum [10]. Because of differences in clinical relevance and species-specific antimicrobial susceptibity and treatment regimens, accurate and rapid species identificaion of SG-NTM is of great importance [7, 9, 11]

Methods

Results

Conclusion