Nontuberculous Mycobacteria (NTM): Microbiological, Clinical and Geographical Distribution

Nontuberculous Mycobacteria (NTM)

Microbiological, Clinical and Geographical Distribution

Edited by

Ali Akbar Velayati

Mycobacteriology Research Centre (MRC), National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran

Parissa Farnia

Mycobacteriology Research Centre (MRC), National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran

Table of Contents

Cover image

Title page

Copyright

List of Contributors

Preface

Chapter 1. The Taxonomy of the Genus Mycobacterium

Abstract

References

Chapter 2. Identification of Nontuberculous Mycobacterium: Conventional Versus Rapid Molecular Tests

Abstract

Species Concept in Mycobacterium

Laboratory Procedures for Mycobacterium Identification

Thiophen-2-Carboxylic Acid Hydrazide

Three- and Fourteen-Day Arylsulfatase Test

Oxygen Preference Test

Genomic DNA Extraction from Mycobacterial Culture

References

Further Reading

Chapter 3. Susceptibility Testing of Nontuberculous Mycobacteria

Abstract

Determination of Clinical Significance

Mechanisms of Resistance

Phenotypic Drug Susceptibility Testing for Nontuberculous Mycobacteria

Genotypic Drug Susceptibility Testing for Nontuberculous Mycobacteria

Treatment of Nontuberculous Mycobacteria Infections

Conclusion

References

Chapter 4. Future Nontuberculous Mycobacteria DST and Therapeutic Interventions

Abstract

Current Challenges of Drug Susceptibility Testing

The Role of Testing Drug Combination Therapy

Future Therapeutic Approaches

Synergistic Treatment Regimens

Potential New Treatments

Inhalation Treatment Regimens

Intermittent Treatment Regimens

Therapeutic Vaccine Approaches

Summary

References

Chapter 5. Nontuberculous Mycobacterial Diseases in Humans

Abstract

Etiological Agents

Epidemiology and Transmission

Pathogenesis and Immunity

Predisposing Factors for Nontuberculous Mycobacteria Diseases

Bacteriological Diagnosis

Clinical Diagnosis and Presentation of Nontuberculous Mycobacteria Disease in Children

Clinical Diagnosis and Presentation of Nontuberculous Mycobacteria Disease in Adults

Therapy for Nontuberculous Mycobacteria Infections in Children

Therapy for Nontuberculous Mycobacteria Infections in Adults

Prognosis

Conclusions

References

Chapter 6. Nontuberculous Mycobacterial Lung Disease

Abstract

Introduction

Clinical Manifestations of Nontuberculous Mycobacteria Lung Disease

Chronic Obstructive Airways Disease, Bronchiectasis, and Nontuberculous Mycobacteria

Nontuberculous Mycobacteria in Patients With Cystic Fibrosis

Nontuberculous Mycobacteria in Immunocompromised Patients

Summary and Conclusions

References

Chapter 7. Clinical Presentation of Nontuberculous Mycobacteria Using Radiological and CT Scan Imagining

Abstract

Introduction

Imaging in Nontuberculous Mycobacteria Group

Lung Infection With Common Rapidly Growing Mycobacteria

Lung Infection With Common Slow Growing Mycobacteria

Conclusion

References

Further Reading

Chapter 8. Mapping the Footprints of Nontuberculous Mycobacteria: A Diagnostic Dilemma

Abstract

Introduction

Microbiological Diagnosis of Nontuberculous Mycobacterial Disease

Conclusions

References

Chapter 9. Nosocomial and Healthcare-Associated NTM Infections and Their Control

Abstract

Epidemiology of Healthcare-Associated Nontuberculous Mycobacterial Infections

Challenges of Identifying and Reporting Healthcare-Associated Nontuberculous Mycobacterial Infections and Outbreaks

Preventive and Control Measures

References

Chapter 10. Epidemiological Distribution of Nontuberculous Mycobacteria Using Geographical Information System

Abstract

Introduction

References

Index

Copyright

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List of Contributors

Imran Ahmed,     Department of Pathology and Laboratory Medicine, Aga Khan University, Karachi, Pakistan

Rutger Bennet,     Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Stockholm, Sweden

Mridula Bose,     Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India

Margareta Eriksson,     Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Stockholm, Sweden

Parissa Farnia,     Mycobacteriology Research Centre (MRC), National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran

Poopak Farnia,     Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Jalaledin Ghanavi,     Mycobacteriology Research Centre (MRC), National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran

Rumina Hasan,     Department of Pathology and Laboratory Medicine, Aga Khan University, Karachi, Pakistan

Sven Hoffner,     Department of Public Health Sciences, Karolinska Institute, Stockholm, Sweden

Seema Irfan,     Department of Pathology and Laboratory Medicine, Aga Khan University, Karachi, Pakistan

Hamidreza Jamaati,     Chronic Respiratory Diseases Research Center, National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran

Bodil Jönsson,     Department of Infectious Medicine, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden

Lars-Olof. Larsson,     Division of Respiratory Medicine, Department of Medicine, Karolinska University Hospital, Stockholm, Sweden

Payam Mehrian,     Pediatric Respiratory Diseases Research Centre (PRDRC), National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran

Diane Ordway,     Mycobacteria Research Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, United States

Maria Owais,     Ziauddin Medical University, Karachi, Pakistan

Malin Ridell,     Department of Microbiology and Immunology, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden

Ruxana T. Sadikot,     Section of Pulmonary, Critical Care Medicine, Emory University, Atlanta, GA, United States

Shima Saif,     Mycobacteriology Research Centre (MRC), National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran

Sadia Shakoor,     Department of Pathology and Laboratory Medicine, Aga Khan University, Karachi, Pakistan

Payam Tabarsi,     Clinical Tuberculosis and Epidemiology Research Center, National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran

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

Mandira Varma-Basil,     Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India

Ali Akbar Velayati,     Mycobacteriology Research Centre (MRC), National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran

Preface

Ali Akbar Velayati and Parissa Farnia, Mycobacteriology Research Centre (MRC), National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran

There has been a recent dramatic interest in the incidence of pulmonary nontuberculous Mycobacteria (NTM). Twenty years ago, NTM were regarded as nonvirulent bacteria that were not appreciated widely as human pathogens. Today, they are considered to be in every environmental factor, that is, soil, water, and air. Recently, it was shown that NTM can cause diseases from various environmental conditions rather than from person-to-person in very rare exceptional cases. Ernest Runyon (1959) put these human disease-associated Mycobacteria into four main groups. The number of identified and cataloged NTM species has been increasing rapidly, from about 50 in 1997 to over 160 by January, 2016. The surge is mainly due to improved isolation and identification techniques. However, even with these new techniques, the Runyon classification is still sometimes used to organize the Mycobacteria into categories. The most common clinical manifestation of NTM disease is lung disease, but lymphatic, skin/soft tissue, and disseminated diseases are also important. Pulmonary disease caused by NTM is most often seen in postmenopausal women and patients with underlying lung disease such as cystic fibrosis (CF), bronchiectasis, and prior tuberculosis. It is not uncommon for alpha 1-antitrypsin deficiency, Marfan syndrome, or primary ciliary dyskinesia patients to have pulmonary NTM colonization and/or infection. Pulmonary NTM can also be found in individuals with AIDS and malignant disease. It can be caused by many NTM species which depends on region, but most frequently MAC and M. kansasii. Clinical symptoms vary in scope and intensity, but commonly include chronic cough, often with purulent sputum. Hemoptysis may also be present. Systemic symptoms include malaise, fatigue, and weight loss in advanced stages of disease. The diagnosis of NTM pulmonary infection requires the presence of symptoms, radiologic abnormalities, and microbiologic cultures. In this book, we give an overview of the taxonomy and identification of NTM. Then, the susceptibility of these species are discussed. Additionally to clinical symptoms and radiological patterns, the geographical distribution of NTM are presented. The authors hope that by going through the chapters of this book, clinicians and researchers will give more attention to NTM. Although one should keep in mind that NTM study needs intellectual trends, funds, and governmental support for high tech-laboratories to detect and report NTM infection within suspected cases.

Reference

1. Runyon EH. Anonymous mycobacteria in pulmonary disease. Med Clin North Am. 1959;43(1):273–290 PubMed 3612432.

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 helix 18 as well.

• The long helix 18 (12-nucleotide insertion) is a marker of slow growers.

• A small group of slow growers related to M. terrae (M. terrae complex) have an even longer helix 18 (14-nucleotide insertion).

M. doricum and M. tusciae where regarded as the exceptions to the theorem above.

In the first decade of the 21st century, with the objective of improving the robustness of the phylogenetic trees, concatenated sequences of a few housekeeping genes were used for inference (Devulder et al., 2005; Mignard and Flandrois, 2008; Tortoli, 2012). The reduction of the weight of 16S rRNA, due to the presence of other genes in the concatenated sequence, was expected to minimize or even to sponge out the theory of the signatures. This was not the case, the 16S signatures continued to predict the topology of the phylogenetic supertree. M. doricum and M. tusciae were confirmed as the only exceptions.

The ultimate taxonomy of the genus Mycobacterium is being written right now. So far, two studies based on whole genomes have dealt with the phylogeny of the genus Mycobacterium (Fedrizzi et al., 2017; Tortoli et al., 2017). The number of genomes were different, as well as the approaches. Trees were reconstructed starting from the core genomes (concatenated sequences of the genes shared by all strains), the gene presence/absence clustering (Fedrizzi et al., 2017), and the average nucleotide identity (ANI) (Tortoli et al., 2017). The first tree was inferred from concatenated sequences using the maximum likelihood (ML) algorithm; the second was obtained, again with the ML algorithm, after converting the clusters to a binary matrix encoding the presence of each strain in the clusters; the third was constructed from the distance matrix of pairwise ANI-divergences using the ML, the neighbor-joining and the unweighted pair group method using arithmetic averages (UPGMA) algorithms. The similarity between topologies of different trees is striking.

The most ancestral branch leads to the species of the M. abscessus-chelonae complex. The other rapid growers emerge from a more recent branch giving rise to a major clade, accommodating the species of the M. fortuitum-smegmatis group, and a large number of dispersed species. Other progressively more recent branches lead to the M. terrae complex and to slow growers. Among slow growers several clusters of species are present. Two minor close groupings include the species related to M. xenopi and M. celatum. A large cluster includes the major pathogens (M. tuberculosis, M. leprae, M. ulcerans) and a number of species frequently involved in human diseases. Other clusters include the species of the M. simiae complex and the M. avium complex. Most of the 16S rRNA signatures are confirmed, only the one related to the M. simiae complex emerges poorly specific as part of the species presenting the signature do not cluster with M. simiae, but are dispersed among other slow growers not part of the complex. Interestingly, M. doricum and M. tusciae cluster again with rapid growers (Figs. 1.4 and 1.5).

Figure 1.4 Phylogenetic tree of slow growers (excluding M. terrae complex) inferred with the maximum likelihood algorithm on 10,878 ANI-divergence scores (Tortoli, 2017), bootstrapped 500 times. The tree was rooted with Hoyosella altamirensis as outgroup. Bar, 2 units difference of ANI-divergence values.

Figure 1.5 Phylogenetic tree of M. terrae complex and rapid growers inferred with the maximum likelihood algorithm on 10,878 ANI-divergence scores (Tortoli, 2017), bootstrapped 500 times. The tree was rooted with Hoyosella altamirensis as outgroup. Bar, 2 units difference of ANI-divergence values.

Several major traits characterize the phylogenetic scenario emerging from whole genome data. Primordial mycobacteria were rapid growers more closely related to the M. abscessus-chelonae complex. The evolution toward slow growers, likely associated with the acquisition of a 14-nucleotide insertion in the helix 18, leads to the species related to the present M. terrae complex. The deletion of two nucleotides in the helix 18 made the 12-nucleotide insertion the marker of the most successful slow growers. In contrast with previous knowledge, the lost of the whole 12-nucleotide insertion is not a specific marker of M. simiae complex. It is actually shared by a number of paraphyletic species.

Interestingly, the present pathogenic species and most of those frequently responsible of opportunistic diseases in humans appear to have evolved from a common ancestor. The latter hypothesis is not new, it was in fact hypothesized on the basis of the presence, in many such species, of genes related with virulence (van Ingen et al., 2012).

A recent paper (Gupta et al., 2018), published after the completion of present review, redraws in deep the classification of mycobacteria. The Authors split the genus Mycobacterium in five genera basing on molecular markers consisting, either in insertions/deletions of amino acids, or in proteins exclusively found in evolutionarily-related groups of species. Far from distorting the aforesaid phylogenetic reconstruction, the new genera exactly overlap the major clades of classical taxonomy: M. abscessus complex, other rapid growers, M. terrae complex and M. triviale-related species.

References

1. Barbu E, Lee KY, Wahl R. Contenu en bases puriqniue et pyrimidiniques des acides deoxyribonucleiques bacteriques. Ann Inst Pasteur (Paris). 1956;91:212–224.

2. Böddinghaus B, Rogall T, Flohr T, Blöcker H, Böttger EC. Detection and identification of mycobacteria by amplification of rRNA. J Clin Microbiol. 1990;28:1751–1759.

3. Brennan PJ, Nikaido H. The envelope of mycobacteria. Annu Rev Biochem. 1995;64:29–63.

4. Chan JZ, Halachev MR, Loman NJ, Constantinidou C, Pallen MJ. Defining bacterial species in the genomic era: insights from the genus Acinetobacter. BMC Microbiol. 2012;12:302.

5. Devulder G, Perouse DM, Flandrois JP. A multigene approach to phylogenetic analysis using the genus Mycobacterium as a model. Int J Syst Evol Microbiol. 2005;55:293–302.

6. Fedrizzi T, Meehan CJ, Grottola A, et al. Genomic characterization of nontuberculous mycobacteria. Sci Rep. 2017;7:45258.

7. Gevers D, Cohan FM, Lawrence JG, et al. Opinion: re-evaluating prokaryotic species. Nat Rev Microbiol. 2005;3:733–739.

8. Gupta RS, Lo B, Son J. Phylogenomics and comparative genomic studies robustly support division of the genus Mycobacterium into an emended genus Mycobacterium and four novel genera. Front Microbiol. 2018;9:67.

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12. Rogall T, Wolters J, Flohr T, Böttger EC. Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium. Int J Syst Bacteriol. 1990;40:323–330.

13. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U.S.A. 1977;74:5463–5467.

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17. Tortoli E. Phylogeny of the genus Mycobacterium: many doubts, few certainties. Infect., Genet Evol. 2012;12:827–831.

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19. van Ingen J, Boeree MJ, van Soolingen D, Iseman MD, Heifets LB, Daley CL. Are phylogenetic position, virulence, drug susceptibility and in vivo response to treatment in mycobacteria interrelated?. Infect Genet Evol. 2012;12:832–837.

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Chapter 2

Identification of Nontuberculous Mycobacterium

Conventional Versus Rapid Molecular Tests

Ali Akbar Velayati, Parissa Farnia and Shima Saif,    Mycobacteriology Research Centre (MRC), National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran

Abstract

The main aim of this chapter is to describe the informative steps to diagnose Mycobacterium other than tuberculosis (NTM). Although, the death rate from NTM may not be as high as tuberculosis and lepromatous diseases, it was described that the laboratory work engaged in mycobacteriology should follow the standard classification of methods to detect NTM. The information provided here are the standard protocols recommended by the European Society of Mycobacteriology, the American Society of Microbiology, and the World Health Organization.

Keywords

Identification tests; NTM; classical; molecular

Species Concept in Mycobacterium

Mycobacteria represent a very ancient genus of bacteria.