Mycobacteriology
Standardization of multilocus sequence typing scheme for Mycobacterium abscessus and Mycobacterium massiliense,☆☆

https://doi.org/10.1016/j.diagmicrobio.2013.06.023Get rights and content

Abstract

This study aims to develop a multilocus sequence typing (MLST) scheme for Mycobacterium abscessus complex for the typing of stains within each species. A total of 89 clinical isolates of M. abscessus complex from 71 patients of 2 tertiary care hospitals in South Korea were included. Forty-two isolates were identified as M. abscessus, and 29, as Mycobacterium massiliense through sequencing of 8 housekeeping genes and rpoB. The MLST scheme identified 26 different sequence types(STs) and 13 different clonal complexes (CCs) in M. abscessus and 12 different STs and 6 different CCs in M. massiliense. The MLST data showed high concordance with the XbaI-macrorestriction patterns of pulsed-field gel electrophoresis in the duplicated isolates. Our MLST schemes could identify different strains of M. abscessus and M. massiliense, and the schemes also showed a reliable reproducibility. Therefore, our MLST schemes may be useful in studying the epidemiology of M. abscessus and M. massiliense infections.

Introduction

Mycobacterium abscessus complex is a rapid-growing mycobacterium (RGM) that causes a wide spectrum of diseases in humans (Anonymous, 1997, Griffith et al., 2007). It accounts for 65–80% of RGM lung diseases (Griffith et al., 1993, Han et al., 2007) and is the second common RGM species causing extrapulmonary diseases following Mycobacterium fortuitum (Daley and Griffith, 2002, Griffith et al., 1993, Griffith et al., 2007). Recent studies revealed that M. abscessus complex comprises 3 closely related species: M. abscessus, Mycobacterium massiliense, and Mycobacterium bolletii (Adékambi et al., 2004, Adékambi et al., 2006). Diverse methods, including partial rpoB gene sequencing (Adékambi and Drancourt, 2004, Adékambi et al., 2003, Devulder et al., 2005, Mollet et al., 1997, Shin et al., 2008), polymerase chain reaction (PCR) restriction enzyme analysis of the hsp65 gene (PRA-hsp65), restriction fragment length polymorphims (RFLP) analysis of the 16S rRNA gene, DNA-DNA hybridization (Leao et al., 2009), pulsed-field gel electrophoresis (PFGE), and multilocus sequence analysis (MLSA), have been tried to be used for the species identification of M. abscessus complex (Adékambi and Drancourt, 2004, Macheras et al., 2009, Rubin et al., 2003).

Non-tuberculous mycobacteria (NTM), including the M. abscessus complex, are distributed in the soil, natural water, and treated water sources of the environment (Falkinham, 2002). Typically, the specific source of NTM human infections cannot be identified, although NTM human diseases are suspected to be acquired from environmental exposure (von Reyn et al., 2002). There is currently no evidence of human-to-human transmission of NTM, even in highly susceptible populations (Guerrero et al., 1995, Meissner and Anz, 1977, Olivier et al., 2003, Tanaka et al., 2000, von Reyn et al., 1993). Strain typing of the M. abscessus complex is important for understanding the epidemiology of infections. Such information is critical for 1) the identification of the sources of M. abscessus complex in patients with primary infection, 2) the differentiation between reactivation and reinfection by another strain in patients with recurred infection, and 3) the analysis of outbreaks of infection caused by the M. abscessus complex (Koh et al., 2010, Tiwari et al., 2003).

Previously described methods are limited in their ability to determine M. abscessus complex strain types. First, methods such as partial rpoB gene sequencing, PRA-hsp65 RFLP analysis of the 16S rRNA gene, and DNA-DNA hybridization (Leao et al., 2009, Macheras et al., 2009) are unable to differentiate some closely related species like the M. abscessus complex in spite of attempt. Second, MLSA can be used only in species identification and not strain typing. Third, PFGE, which has been regarded as the gold standard technique for epidemiologic studies of NTM, shows poor inter-laboratory reproducibility (Wallace et al., 1998).

Multilocus sequence typing (MLST) is a PCR and sequencing-based technique to identify sequence types (STs) of strains by compounding the allelic types of several housekeeping genes (Enright and Spratt, 1999). The method is now available and widely used for identifying STs of strains in many bacterial species, including Staphylococcus aureus (Enright et al., 2000), Streptococcus pneumoniae (Feil et al., 2000), Enterococcus faecium (Homan et al., 2002), Klebsiella pneumoniae (Giske et al., 2012), Acinetobacter baumannii (Bartual et al., 2005), and Pseudomonas aeruginosa (Curran et al., 2004). A major advantage of MLST is the possibility of data exchange and comparison through an electronic platform due to using of nucelotide sequence data. However, an MLST scheme for M. abscessus complex has yet to be developed, although that will be useful for understanding the epidemiology of infections with species of this complex.

In the present study, we developed an MLST scheme to type the strains within each species of the M. abscessus complex.

Section snippets

Setting and bacterial strains

This study included a total of 89 clinical M. abscessus complex isolates from 71 patients of 2 tertiary care hospitals (Severance Hospital and Seoul National University Hospital) in Seoul, Republic of Korea, between January 2011 and August 2011. Duplicate isolates were obtained from 16 patients (interval: mean, 2.5 months; range, 0–9.6 months). All isolates were recovered from respiratory specimens of patients with M. abscessus complex lung diseases. All patients met the American Thoracic

Identification of the M. abscessus complex by MLSA and rpoB sequencing

Phylogenetic trees were obtained from the partial sequences of 8 housekeeping genes of MLSA and rpoB. The trees showed that our collection of isolates comprised 2 principal groups (MLSA: group A, n = 42; group B, n = 29; rpo sequencing: group A′, n = 43; group B′, n = 28), and each group included type strain M. abscessus CIP104536T and M. massiliense CIP108297T, respectively. Therefore, the groups were assigned as M. abscessus and M. massiliense, respectively. The identification of strains

Discussion

We identified species of M. abscessus complex using MLSA and rpoB before developing the MLST scheme. There was a discrepancy in the identification of M. abscessus complex between MLSA and rpoB sequencing in only a single isolate (strain 6). This isolate was identified as M. massiliense by MLSA but as M. abscessus by rpoB sequencing. The isolate grouped as a different cluster from other clinical M. massiliense isolates in the phylogenetic tree generated using the concatenated sequences of 7 MLST

Acknowledgments

This work was supported by a National Research Foundation of Korea grant funded by the Korean Government (2011-0013018).

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