Nontuberculous Mycobacteria (NTM)

Microbiological, Clinical and Geographical Distribution

Ali Akbar Velayati, Parissa Farnia

• Publisher: Academic Press
• Year: 2019
• ISBN:
  9780128146934
• Topic: Biological Sciences
• Subtopic: Biology
• Index: Biological Sciences

Nontuberculous Mycobacteria (NTM): Microbiological, Clinical and Geographical Distribution is a complete reference that stimulates a greater understanding of NTM infections. Sections cover microbiologic and molecular diagnostic tools, drug susceptibility tests, human genetic susceptibility, prevalence and incidence studies, clinical and radiological presentations, and clinical trials for antibiotic therapy. With the incidence rate of NTM infections increasing globally during the last decade, significant mortality and morbidity must be addressed. This important reference will provide research scientists, clinical microbiologists, hospital diagnostic technicians, and post graduate medical and science students with information on the epidemiology, prevalence, microbiology and clinical aspects of NTM.

• Highlights new findings in the epidemiological distribution and new diagnosis and treatment protocol of mycobacterial infections
• Debates new advances in the detection of NTM
• Demonstrates the distribution of NTM in the environment and its relationship with human infection using a geographical information system (GIS)
• Includes new radiological findings in non-tuberculous mycobacterial infections in the lung using CT and PET-Scan imaging

Chapter 1

The Taxonomy of the Genus Mycobacterium

Enrico Tortoli, Emerging Bacterial Pathogens Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy

Abstract

Phylogenetic studies of the genus Mycobacterium have been based, in the past three decades, on the sequence of the 16S rRNA gene, on concatenated sequences of a few housekeeping genes and, very recently, on whole genome sequences. Surprisingly these approaches produced highly consistent phylogenetic reconstructions. In all cases, the rapidly and slowly growing species were clearly separated. The species composition of the major clusters was largely overlapping and one of them, the M. terrae complex, constantly took its place between the branches leading to rapid and slow growers. The clade of rapid growers including the species of the M. abscessus-chelonae complex was revealed to be the most ancestral and is located close to the root of the genus. The pathogenic species (M. tuberculosis and M. leprae) clustered together with most species frequently involved in opportunistic human infections. The major discrepancies among different phylogenetic approaches occurred with a group of species genetically related to M. simiae; the large grouping defined by the 16S sequences emerged scaled down in the phylogeny inferred by the whole genome sequences with most of them reallocated among other, apparently unrelated, species.

Keywords

Phylogeny; mycobacterium; average nucleotide identity; taxonomy; whole genome sequencing; 16S rRNA

The genus Mycobacterium includes more than 190 species and belongs to the family of Mycobacteriaceae, class Corynobacteriales, type Actinobacteria, and kingdom Bacteria. It was first proposed in 1896 (Lehmann and Neuman, 1896) to host organisms considered at that time to be halfway between fungi and bacteria.

The Swedish botanist Carl Linnaeus is considered the founder of taxonomy: a hierarchical classification of plants (Linnaeus, 1735), subsequently extended to animals, based on similarity of phenotypic characters. A similarly important innovation introduced by Linnaeus is the binomial nomenclature, still in use for naming species.

Following the discovery of DNA (Watson and Crick, 1953), genetic characters started to be investigated and to be used for taxonomic purposes. The determination of DNA base composition (guanine + cytosine%) represented the first step (Barbu et al., 1956). A few years later Wayne et al. proposed to measure the DNA relatedness among strains in a paper aiming to reconcile the competing taxonomic approaches: phenotypic and genotypic (Wayne et al., 1987). He suggested that in members of different species this parameter, measured by DNA–DNA hybridization (DDH), should be lower than 70%. The 70% threshold is still considered a gold standard for species circumscription, the DDH test is, however, hardly performed in modern laboratories (Chan et al., 2012).

The genetic sequencing introduced by Sanger (Sanger et al., 1977), a milestone in biological sciences, had an enormous impact on taxonomy and represented significant progress toward a phylogeny-based classification. The rRNA, which is highly conserved because of the essential role of ribosomes in the protein synthesis, soon became the primary target. Among its three subunits, the 16S has been by far the most frequently investigated, and at present the sequence of this locus is available, for every known species, in public databases.

Comparative studies between 16S rRNA sequence and DDH lead to identify a 16S similarity of 97% as the threshold corresponding to 70% DDH (Gevers et al., 2005). Despite the cutoff being subsequently revised and raised to 98.8%–99% (Stackebrandt and Ebers, 2006) it still remains unsuitable to appraise the divergence between several species within the genus Mycobacterium.

The genus Mycobacterium is characterized, at phenotypic level, by unique characteristics. In the cell wall, extremely rich in lipids, the mycolic acids play a major role. Although they are present in a few other Actinobacteria-related genera, only the genus Mycobacterium has chains as long as 60–90 C (Brennan and Nikaido, 1995). On the basis of growth rate, the mycobacteria are conventionally divided into two groups: the slow growers require more than 1 week to develop visible colonies on solid media, while the rapid growers may require 3–7 days, thus growing slower in comparison with other cultivable bacteria (Tortoli, 2003).

Genetically, the mycobacteria differ from the large majority of bacteria for the high G + C content (ranging from 62% to 70%). The number of copies of the ribosomal operon is low: two copies in the rapid growers and only one in the slow growers; there are very few exceptions (Böddinghaus et al., 1990).

The variability of 16S rRNA is moderate among mycobacteria with a number of species presenting 100% identical sequence (Tortoli, 2003). The 16S rRNA is about 1500 bp long and includes mostly highly conserved regions with few interposed variable traits. The two major variable strings are located in the first third of the gene; they are known as hypervariable regions A (E. coli positions 130–210) and B (E. coli positions 430–500) with the latter including the helix 18. Minor variable regions are located in the remaining two thirds of the gene (Stahl and Urbance, 1990). The hypervariable region A hosts most of the species-specific polymorphisms. The hypervariable region B is characterized by three mayor formats; it may host, in the helix 18, a 14-nucleotide insertion, a 12-nucleotide insertion, or no insertion at all.

Since the first taxonomic analyses of the genus Mycobacterium based on the sequence of 16S rRNA, a number of features have emerged. Rapid and slow growers appeared clearly separated in the phylogenetic tree. All the rapidly growing species had a short helix 18 (no insertion in the hypervariable region B). The majority of slow growing species had a long helix 18: 12-nucleotide insertion in the hypervariable region B. The slow growers M. terrae and M. nonchromogenicum had a 14-nucleotide insertion and were located in a separate clade interposed between slow and rapid growers. M. simiae did not have insertion in the hypervariable region B, but clustered with slow growers according to its phenotype (Rogall et al., 1990; Stahl and Urbance, 1990) (Fig. 1.1).

Figure 1.1 Phylogenetic tree inferred with the neighbor-joining algorithm from the sequence dataset investigated by Rogall et al. (1990). The tree was rooted with Nocardia asteroids as outgroup. Bar, 0.005 substitutions per nucleotide position.

In subsequent years, the inclusion in the analysis of further species did not produce substantial changes in the topology of the phylogenetic tree. Among the newly added species, the new rapid growers clustered with the previous ones and confirmed the short helix 18; the species which added to the clade of M. terrae and M. nonchromogenicum had the 14-nucleotide insertion; most of the slow growers confirmed the presence of a 12-nucleotide insertion. The few slow growers with short helix 18 grouped with M. simiae except two, M. doricum and M. tusciae, which fell among the rapid growers (Figs. 1.2 and 1.3).

Figure 1.2 Phylogenetic tree of slow growers inferred with the neighbor-joining algorithm from the sequence dataset investigated by Tortoli (2012), bootstrapped 500 times. The tree was rooted with Nocardia asteroides as outgroup. Bar, 0.005 substitutions per nucleotide position.

Figure 1.3 Phylogenetic tree of rapid growers inferred with the neighbor-joining algorithm from the sequence dataset investigated by Tortoli (2012), bootstrapped 500 times. The tree was rooted with Nocardia asteroides as outgroup. Bar, 0.005 substitutions per nucleotide position.

On the basis of such results, the presence of signatures within the 16S rRNA was theorized:

• • The short helix 18 is a marker of rapid growers.
• • A small group of slow growers related to M. simiae (M. simiae complex), recognizable for sharing the same sequence in the hypervariable region B, have the short...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• List of Contributors
• Preface
• Chapter 1. The Taxonomy of the Genus Mycobacterium
• Chapter 2. Identification of Nontuberculous Mycobacterium: Conventional Versus Rapid Molecular Tests
• Chapter 3. Susceptibility Testing of Nontuberculous Mycobacteria
• Chapter 4. Future Nontuberculous Mycobacteria DST and Therapeutic Interventions
• Chapter 5. Nontuberculous Mycobacterial Diseases in Humans
• Chapter 6. Nontuberculous Mycobacterial Lung Disease
• Chapter 7. Clinical Presentation of Nontuberculous Mycobacteria Using Radiological and CT Scan Imagining
• Chapter 8. Mapping the Footprints of Nontuberculous Mycobacteria: A Diagnostic Dilemma
• Chapter 9. Nosocomial and Healthcare-Associated NTM Infections and Their Control
• Chapter 10. Epidemiological Distribution of Nontuberculous Mycobacteria Using Geographical Information System
• Index