A hypervariable genomic island identified in clinical and environmental Mycobacterium avium subsp. hominissuis isolates from Germany

https://doi.org/10.1016/j.ijmm.2016.07.001Get rights and content

Abstract

Mycobacterium avium subsp. hominissuis (MAH) is an opportunistic human pathogen widespread in the environment. Genomic islands (GI)s represent a part of the accessory genome of bacteria and influence virulence, drug-resistance or fitness and trigger bacterial evolution. We previously identified a novel GI in four MAH genomes. Here, we further explored this GI in a larger collection of MAH isolates from Germany (n = 41), including 20 clinical and 21 environmental isolates. Based on comparative whole genome analysis, we detected this GI in 39/41 (95.1%) isolates. Although all these GIs integrated in the same insertion hotspot, there is high variability in the genetic structure of this GI: eight different types of GI have been identified, designated A–H (sized 6.2–73.3 kb). These GIs were arranged as single GI (23/41, 56.1%), combination of two different GIs (14/41, 34.1%) or combination of three different GIs (2/41, 4.9%) in the insertion hotspot. Moreover, two GI types shared more than 80% sequence identity with sequences of M. canettii, responsible for Tuberculosis. A total of 253 different genes were identified in all GIs, among which the previously documented virulence-related genes mmpL10 and mce. The diversity of the GI and the sequence similarity with other mycobacteria suggests cross-species transfer, involving also highly pathogenic species. Shuffling of potential virulence genes such as mmpL10 via this GI may create new pathogens that can cause future outbreaks.

Introduction

Non-tuberculous mycobacteria (NTM) are gaining growing attention because the incidence of infections increased in many parts of the world, such as Germany (Ringshausen et al., 2013), US (Adjemian et al., 2012), Japan (Morimoto et al., 2014), Ontario (Canada) (Marras et al., 2013), UK (Moore et al., 2010), the Netherlands (van Ingen et al., 2009) and Queensland (Australia) (Thomson and NTM working group at Queensland TB Control Centre and Queensland Mycobacterial Reference Laboratory, 2010). Due to the high level of antibiotic resistance, NTM infections are difficult to treat (van Ingen et al., 2012). Among more than 150 NTM species, M. avium subsp. hominissuis (MAH) is one of the most clinically relevant for humans (Tortoli, 2014, Wu and Holland, 2015). MAH can cause pulmonary infections, lymphadenitis in children and disseminated infections, especially in immunocompromised hosts (Despierres et al., 2012, Ichikawa et al., 2009, Rindi and Garzelli, 2014). In addition, MAH is also widespread in the environment. It has been isolated from dust, soil and water (Falkinham, 2013, Lahiri et al., 2014a).

Apart from their core genome, genomic islands (GIs) contribute to the accessory genome of bacteria that can be acquired via horizontal gene transfer (Langille et al., 2010). Compared to the whole genome, GIs display a distinct GC content and are additionally characterized by the presence of transposons, integrases, and flanking DNA direct repeats and t-RNA genes (Ahmed et al., 2008, Che et al., 2014, Langille et al., 2010). GIs play an important role in microorganism evolution since they can influence virulence, metabolism or adaptation (Ahmed et al., 2008, Che et al., 2014, Langille et al., 2010). Three GIs were recently identified in Mycobacterium tuberculosis carrying pathogenicity genes (Xie et al., 2014). Regarding NTM, the GI MmGI-1 has been identified in M. massiliense JCM 15300, with putative function in lipid metabolism (Sekizuka et al., 2014). Moreover, one GI has a putative role in zinc metabolism in M. avium subsp. paratuberculosis (Eckelt et al., 2014).

Research on GIs in MAH is ongoing. In a previous study, a GI of 3.5 kb responsible for the mycobacterial invasion of macrophages and amoeba was detected in MAH (Danelishvili et al., 2007). We have recently identified a novel GI in four MAH genomes (Lahiri et al., 2014b). Here, we further explore this GI in over forty clinical and environmental MAH isolates from Germany. We aimed to investigate the presence of this GI in MAH isolated from different sources such as clinical and environmental sources.

Since different GI types and combinations were observed, always integrated in the same insertion hotspot in the genome, we named this GI of MAH “hypervariable GI”.

Section snippets

Bacterial isolates and whole genome sequencing

A total of 41 MAH isolates from clinical or environmental origins from Germany were analyzed, including the two reference strains MAH 104 and MAH TH135. Clinical isolates were provided by the National Reference Center for Mycobacteria (Borstel, Germany) or were isolated from respiratory samples of patients with Cystic Fibrosis obtained from the Charité Hospital (Berlin, Germany). Details on the isolates are given in Table 1. The environmental isolates and their isolation procedure have been

Whole genome sequencing and identification of the GI in 41 MAH isolates

The genome sequence of the reference strains (MAH 104 and MAH TH135) and two German isolates (MAH 2721 and MAH 27-1) were already present in the GenBank database. Here we deposited the whole genome sequences of the remaining 37 isolates at DDBJ/EMBL/GenBank under the BioProject Number PRJNA299461. Table 1 shows the characteristics of the 41 MAH isolates. Genome metrics are shown in Table S1 (see Table S1).

Overall, the GI could be identified in 39/41 isolates (95.1%). Among them, 18 were

Discussion

In this study, we explored in-depth a recently identified GI from MAH in over forty clinical or environmental MAH isolates from Germany.

Researchers have previously identified different GIs in NTM (Eckelt et al., 2014, Sekizuka et al., 2014). However, all these previous studies had been performed on single isolates. This raised the question whether these GIs are isolate-specific, species-specific or do they vary across the species. Our data showed that the recently identified GI from MAH widely

Acknowledgments

We thank Elvira Richter (National Reference Center for Mycobacteria, Borstel, Germany) and Roland Schulze-Röbbecke (University Hospital Düsseldorf) for providing part of the MAH isolates, Carsten Schwarz (Christiane Herzog Zentrum, Charité, Berlin) for providing respiratory samples from cystic fibrosis patients and Kei-ichi Uchiya for the reference strain MAH TH135. We thank Inga Eichorn for the help with the generation of the whole genome sequencing data. We thank Ursula Erikli for the English

References (49)

  • K. Andries et al.

    Acquired resistance of Mycobacterium tuberculosis to bedaquiline

    PLoS One

    (2014)
  • S. Arruda et al.

    Cloning of an M. tuberculosis DNA fragment associated with entry and survival inside cells

    Science

    (1993)
  • R.K. Aziz et al.

    The RAST Server: rapid annotations using subsystems technology

    BMC Genomics

    (2008)
  • T. Bartpho et al.

    Genomic islands as a marker to differentiate between clinical and environmental Burkholderia pseudomallei

    PLoS One

    (2012)
  • J. Becq et al.

    Contribution of horizontally acquired genomic islands to the evolution of the tubercle bacilli

    Mol. Biol. Evol.

    (2007)
  • D. Che et al.

    Identifying pathogenicity islands in bacterial pathogenomics using computational approaches

    Pathogens

    (2014)
  • L. Danelishvili et al.

    Identification of Mycobacterium avium pathogenicity island important for macrophage and amoeba infection

    Proc. Natl. Acad. Sci. U. S. A.

    (2007)
  • L. Despierres et al.

    Diversity of Mycobacterium avium subsp. hominissuis mycobacteria causing lymphadenitis

    France Eur. J. Clin. Microbiol. Infect. Dis.

    (2012)
  • P. Domenech et al.

    Contribution of the Mycobacterium tuberculosis MmpL protein family to virulence and drug resistance

    Infect. Immun.

    (2005)
  • E. Eckelt et al.

    Identification of a lineage specific zinc responsive genomic island in Mycobacterium avium ssp. paratuberculosis

    BMC Genomics

    (2014)
  • J.O. Falkinham

    Ecology of nontuberculous mycobacteria – where do human infections come from?

    Semin. Respir. Crit. Care Med.

    (2013)
  • K. Flikka et al.

    XHM: a system for detection of potential cross hybridizations in DNA microarrays

    BMC Bioinf.

    (2004)
  • M.A. Forrellad et al.

    Virulence factors of the Mycobacterium tuberculosis complex

    Virulence

    (2013)
  • R.C. Hartkoorn et al.

    Cross-resistance between clofazimine and bedaquiline through upregulation of MmpL5 in Mycobacterium tuberculosis

    Antimicrob. Agents Chemother.

    (2014)
  • Cited by (10)

    • Environmental Mycobacterium avium subsp. hominissuis have a higher probability to act as a recipient in conjugation than clinical strains

      2018, Plasmid
      Citation Excerpt :

      Also, genetic differences between clinical and environmental strains are so far not described. In order to define the clinical relevance of this genetic diversity, we have previously conducted a study comparing genomes from clinical and environmental MAH isolates which led to the identification of a new genomic island divided into eight types that considerably adds to the genetic diversity in MAH (Sanchini et al., 2016). Sequence homologies between the genomic islands and DNA from other mycobacterial species suggests that horizontal gene transfer was involved.

    View all citing articles on Scopus
    View full text