# NTM – THE NEW UBER-BUGS

EDITED BY : Veronique Dartois, Christine Sizemore and Thomas Dick PUBLISHED IN : Frontiers in Microbiology

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# NTM – THE NEW UBER-BUGS

Topic Editors:

Veronique Dartois, Center for Discovery and Innovation, Hackensack Meridian Health, United States

Christine Sizemore, National Institute of Allergy and Infectious Diseases, United States

Thomas Dick, Center for Discovery and Innovation, Hackensack Meridian Health, United States

"Mycobacterium colonies" by Michelle Yee

Incidence of pulmonary disease caused by nontuberculous mycobacteria (NTM), such as Mycobacterium abscessus and M. avium, is increasing at an alarming rate, surpassing tuberculosis (TB) in many countries. Treatment durations are extremely long. With low sputum conversion rates, the outcomes are often disappointing. Thus, NTM disease resembles extremely drug resistant TB. There is an urgent need for increased research on this dreadful disease. For this book we brought together experts from a wide range of disciplines addressing current status, gaps and needs, and new developments in several NTM-relevant research areas. We start with the discussion of NTM disease presentations and clinical research. This is followed by contributions on epidemiology, antibiotic resistance mechanisms, drug discovery and development, bacteriology and targets, and new diagnostic tools. We would like to thank the 130 authors for sharing their work and insights. We hope that this collection of articles will stimulate discussions and research activities on the challenging lung disease caused by the diverse group of pathogens called NTM.

Citation: Dartois, V., Sizemore, C., Dick, T., eds. (2019). NTM – The New Uber-Bugs. Lausanne: Frontiers Media. doi: 10.3389/978-2-88963-012-7

# Table of Contents

*06 Editorial: NTM—The New Uber-Bugs* Veronique Dartois, Christine Sizemore and Thomas Dick

#### DISEASE PRESENTATIONS AND CLINICAL RESEARCH

Christopher Vinnard, Alyssa Mezochow, Hannah Oakland, Ross Klingsberg, John Hansen-Flaschen and Keith Hamilton

#### EPIDEMIOLOGY

*28 A Closer Look at the Genomic Variation of Geographically Diverse*  Mycobacterium abscessus *Clones That Cause Human Infection and Disease*

Rebecca M. Davidson

#### RESISTANCE MECHANISMS

#### DRUG DISCOVERY AND DEVELOPMENT

Jimin Zhang, Franziska Leifer, Sasha Rose, Dung Yu Chun, Jill Thaisz, Tracey Herr, Mary Nashed, Jayanthi Joseph, Walter R. Perkins and Keith DiPetrillo

*119 Optimization and Lead Selection of Benzothiazole Amide Analogs Toward a Novel Antimycobacterial Agent*

Mary A. De Groote, Thale C. Jarvis, Christina Wong, James Graham, Teresa Hoang, Casey L. Young, Wendy Ribble, Joshua Day, Wei Li, Mary Jackson, Mercedes Gonzalez-Juarrero, Xicheng Sun and Urs A. Ochsner

*129* Mycobacterium abscessus *and ß-Lactams: Emerging Insights and Potential Opportunities* Elizabeth Story-Roller, Emily C. Maggioncalda, Keira A. Cohen and

Gyanu Lamichhane

*139 Novel Screen to Assess Bactericidal Activity of Compounds Against Non-replicating* Mycobacterium abscessus

Bryan J. Berube, Lina Castro, Dara Russell, Yulia Ovechkina and Tanya Parish

### BACTERIOLOGY AND TARGETS

*179 MmpL3 as a Target for the Treatment of Drug-Resistant Nontuberculous Mycobacterial Infections*

Wei Li, Amira Yazidi, Amitkumar N. Pandya, Pooja Hegde, Weiwei Tong, Vinicius Calado Nogueira de Moura, E. Jeffrey North, Jurgen Sygusch and Mary Jackson

*188 Lsr2 is an Important Determinant of Intracellular Growth and Virulence in*  Mycobacterium abscessus

Vincent Le Moigne, Audrey Bernut, Mélanie Cortès, Albertus Viljoen, Christian Dupont, Alexandre Pawlik, Jean-Louis Gaillard, Fabienne Misguich, Frédéric Crémazy, Laurent Kremer and Jean-Louis Herrmann

*199 Glycopeptidolipids, a Double-Edged Sword of the* Mycobacterium abscessus *Complex*

Ana Victoria Gutiérrez, Albertus Viljoen, Eric Ghigo, Jean-Louis Herrmann and Laurent Kremer

*207* Mycobacterium abscessus *subsp.* massiliense mycma\_0076 *and*  mycma\_0077 *Genes Code for Ferritins That are Modulated by Iron Concentration*

Fábio M. Oliveira, Adeliane C. Da Costa, Victor O. Procopio, Wanius Garcia, Juscemácia N. Araújo, Roosevelt A. Da Silva, Ana Paula Junqueira-Kipnis and André Kipnis

#### DIAGNOSTIC TOOLS

*223 Evaluation of a Novel MALDI Biotyper Algorithm to Distinguish*  Mycobacterium intracellulare *From* Mycobacterium chimaera

L. Elaine Epperson, Markus Timke, Nabeeh A. Hasan, Paul Godo, David Durbin, Niels K. Helstrom, Gongyi Shi, Markus Kostrzewa, Michael Strong and Max Salfinger

#### *229 MALDI Spectra Database for Rapid Discrimination and Subtyping of*  Mycobacterium kansasii

Jayaseelan Murugaiyan, Astrid Lewin, Elisabeth Kamal, Zofia Bakuła, Jakko van Ingen, Vit Ulmann, Miren J. Unzaga Barañano, Joanna Humięcka, Aleksandra Safianowska, Uwe H. Roesler and Tomasz Jagielski

## Editorial: NTM—The New Uber-Bugs

#### Veronique Dartois 1 , Christine Sizemore2 and Thomas Dick 1 \*

1 Center for Discovery and Innovation, Hackensack Meridian Health, Nutley, NJ, United States, 2 Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, United States

Keywords: non-tuberculous mycobacteria, environmental mycobacteria, lung disease, repositioning, from biology to solutions

**Editorial on the Research Topic**

#### **NTM—The New Uber-Bugs**

Pulmonary disease caused by non-tuberculous mycobacteria (NTM), relatives of Mycobacterium tuberculosis, is increasing at an alarming rate, surpassing tuberculosis (TB) in many countries. Patients suffering from chronic pulmonary diseases, including Cystic Fibrosis and Chronic Obstructive Pulmonary Disease, are particularly susceptible to NTM infections. These infections are difficult to diagnose and present significant challenges for treatment. The environmental reservoir of the infectious agents and an incomplete understanding of what makes individuals susceptible to infection complicates preventive approaches. Considering the rising incidence and likely underreporting of these infections, a clear understanding of the barriers to diagnosis, treatment and prevention, and focused research on the pathobiology and microbiology of NTMs and new methods to combat them is urgently needed.

#### Edited by:

Axel Cloeckaert, Institut National de la Recherche Agronomique (INRA), France

#### Reviewed by:

Philippe Lanotte, Université de Tours, France

\*Correspondence: Thomas Dick<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

> Received: 23 April 2019 Accepted: 24 May 2019 Published: 12 June 2019

#### Citation:

Dartois V, Sizemore C and Dick T (2019) Editorial: NTM—The New Uber-Bugs. Front. Microbiol. 10:1299. doi: 10.3389/fmicb.2019.01299

Recognizing this emerging new health threat and the complex nature of these infections, the National Institute of Allergy and Infectious Diseases organized the first NTM-focused workshop "Advancing Translational Science for Pulmonary NTM Infections" in Rockville, MD, in September 2017. This workshop, bringing together scientists and physicians from the United States and abroad, identified gaps in fundamental biological and epidemiological research and mapped out possible approaches for targeted translational science to expedite improvements in detection and care (Daniel-Wayman et al., 2018). The gathering not only generated increased awareness of this new health issue, it also brought together research disciplines and investigators from across the world who previously had few opportunities to interact, and kick-started new collaborative research activities. Momentum created during this workshop also resulted in the Research Topic "NTM—The New Uber-Bugs."

NTM basic and translational research are still in their infancy with many questions remaining to understand the biological diversity of the infecting pathogens, their similarities and differences and how these affect pathobiology, transmission, and response to therapy. Furthermore, depending on where NTM infections occur globally, different species of NTM predominate, making rapid diagnostics that can speciate mycobacteria a necessity. This acute need to reduce the biological uncertainties around NTM pulmonary disease by increasing research efforts reminds of the state of knowledge TB research was facing two decades ago. Despite an initial research void in the TB field during the last quarter of the twentieth century, in part driven by the absence of molecular tools and animal models to study this difficult pathogen, significant progress has been achieved since the early 2000s. Knowledge and tools developed for the study of TB disease and approaches and technologies and platforms that have been applied to TB product development can be leveraged for NTM research.

**6**

For instance, chemical entities with activity against Mycobacterium tuberculosis that have been generated in TB screening campaigns can quickly also be tested against NTM and may facilitate repositioning and repurposing of chemical leads and create a complementary approach to de novo drug discovery. By exploiting strategies and tools developed for TB, together with increasing research and product development focused on high-priority NTM species, we believe that significant medical advances for NTM diseases will be achieved in the medium term.

The present Research Topic, "NTM—The New Uber-Bugs" compiles contemporary results and insights from a group of leading researchers into the high-priority research area of NTM infections. It also highlights that in order to truly understand the diverse nature of these infections and their impact on human health, more thorough trans-national collaborations and data sharing will be important. We would like to thank the reviewers for their many thoughtful and insightful comments, and the authors for their excellent contributions. We hope that this collection of manuscripts will stimulate much-needed discussions and research activities on the challenging lung disease caused by the diverse group of pathogens called nontuberculous mycobacteria.

#### REFERENCES

Daniel-Wayman, S., Abate, G., Barber, D. L., Bermudez, L. E., Coler, R. N., Cynamon, M. H., et al. (2018). Advancing translational science for pulmonary nontuberculous mycobacterial infections. a road map for research. Am. J. Respir. Crit. Care Med. 199, 947–951. doi: 10.1164/rccm.201807-1273PP

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

#### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

#### FUNDING

TD holds a Toh Chin Chye Visiting Professorship at the Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore. His work is supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Numbers R01AI132374 and 2R01AI106398-05 and by the Cystic Fibrosis Foundation under Award Number DICK17XX00. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Cystic Fibrosis Foundation.

#### ACKNOWLEDGMENTS

We are grateful to all the authors and reviewers for their contributions to this Research Topic.

Copyright © 2019 Dartois, Sizemore and Dick. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Host Variability in NTM Disease: Implications for Research Needs

Colin Swenson1 \*, Christa S. Zerbe2 and Kevin Fennelly3

1 Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, Emory University, Atlanta, GA, United States, 2 Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States, 3 Laboratory of Chronic Airway Infection, Pulmonary Branch, Division of Intramural Research, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, United States

Non-tuberculous mycobacteria (NTM) are ubiquitous environmental organisms that may cause opportunistic infections in susceptible hosts. Lung infections in immunocompetent persons with structural lung disease are most common, while disseminated disease occurs primarily in immunocompromised individuals. Human disease caused by certain species, such as Mycobacterium avium complex, Mycobacterium abscessus, and Mycobacterium kansasii, is increasing in incidence and varies by geographic distribution. The spectrum of NTM disease varies widely in presentation and clinical outcome, but certain patterns can be organized into clinical phenotypes. Treatment options are limited, lengthy, and often toxic. The purpose of this case-based review is to provide non-clinician scientists with a better understanding of human NTM disease with an aim to stimulate more research and development.

#### Edited by:

Thomas Dick, Rutgers, The State University of New Jersey, United States

#### Reviewed by:

Jennifer R. Honda, National Jewish Health, United States Mary Ann Degroote, SomaLogic, United States

#### \*Correspondence:

Colin Swenson<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

Received: 06 August 2018 Accepted: 12 November 2018 Published: 03 December 2018

#### Citation:

Swenson C, Zerbe CS and Fennelly K (2018) Host Variability in NTM Disease: Implications for Research Needs. Front. Microbiol. 9:2901. doi: 10.3389/fmicb.2018.02901 Keywords: non-tuberculous mycobacteria, pulmonary, bronchiectasis, disseminated, COPD, cystic fibrosis, osteomyelitis

"It is more important to know what sort of person has a disease than to know what sort of disease a person has."

– Hippocrates, Greek Physician, 460–370 B.C.

### INTRODUCTION

Non-tuberculous mycobacteria (NTM) are ubiquitous environmental organisms that are frequently isolated from soil and water, as well as other environmental sources. NTM are acid-fast bacilli (AFB) that are clinically categorized as either rapid growers (RG), taking fewer than 7 days to grow on culture media, or slow growers (SG), taking more than 7 days (**Table 1**). Certain species are well known causes of human disease, most commonly Mycobacterium avium complex (MAC), which includes the species M. avium, M. intracellulare, and M. chimaera, and the M. abscessus complex (MABSC), which includes the subspecies abscessus, massiliense, and bolletii. Considered opportunistic pathogens, NTM organisms may occasionally be isolated from the sputa samples of healthy individuals, but there is a higher prevalence of NTM infection among patients with cystic fibrosis and non-cystic fibrosis bronchiectasis, as well as chronic obstructive pulmonary disease (COPD) (Adjemian et al., 2012; Fleshner et al., 2016). While all MAC species may be isolated from water and soil sources, M. avium is more commonly isolated from fresh water sources, and M. intracellulare is associated with garden soils (Falkinham, 2015). This predilection has been proposed as an explanation for the epidemiologic clusters observed in regions with abundant surface water and loamy soil.

TABLE 1 | Clinically important mycobacterial species organized by growth time.

Slow growing organisms take greater than 7 days to culture. Rapid growing organisms take fewer than 7 days to culture. ∗ Includes M. avium, M. intracellulare, and M. chimaera. ∗∗Includes M. abscessus, M. massiliense, and M. bolletii.

While NTM species may rarely cause pulmonary infections in normal hosts, those with structural lung disease, such as bronchiectasis and emphysema, are at increased risk. The hallmark of the disease is granulomatous inflammation of the airways, which, over time, may lead to worsening bronchiectasis and cavitary lung disease (Fujita et al., 1999). Among patients with pulmonary NTM infections, distinct phenotypes have been described based primarily on the underlying lung disease. These phenotypes have characteristic presentations on lung imaging (**Table 2**).

Although once considered rare diseases, human infections with NTM appear to be increasing in North America and Europe, and they are increasingly recognized in areas of the world considered endemic for tuberculosis (TB), including Africa, South America and India (Adjemian et al., 2012). Human lung disease caused by NTM was first recognized in the 1950s as the incidence of TB was decreasing (Timpe and Runyon, 1954). Given the similarities of clinical microbiology and of the drugs used to treat TB, the care of patients with NTM disease has often been provided by clinicians and hospitals initially established to manage TB. The HIV/AIDS epidemic introduced many physicians and scientists to disseminated infections with MAC, which has waned in incidence along with other opportunistic infections due to the development and widespread use of highly active antiretroviral therapy. Most disseminated NTM infections occur in immunocompromised patients. Similar to TB, pulmonary NTM disease accounts for approximately 85% of cases and disseminated NTM disease for about 15% (Jones et al., 2018). The major types of human NTM disease are summarized in **Table 3**.

The primary purpose of this review is to introduce microbiologists and other scientists who are not clinicians to the wide variety of clinical presentations among humans infected with NTM. Given the variable responses observed to infections with the same pathogen among various inbred strains of mice and other experimental animal models, it should not be surprising that the 'human host' is not just one host, but rather many. However, in spite of the enormous genetic variability among humans, there are similarities in presentations of NTM disease among various human 'phenotypes.' We will present representative cases, most of which are actual cases that we have seen, followed by a discussion of that category of disease and figures of imaging studies. We hope that a better understanding of the various human diseases caused by NTM can help inspire our

FIGURE 1 | Axial CT chest image demonstrating bronchiectasis and mucoid impaction in the right middle lobe. The tree-in-bud nodularity in the left lung (yellow arrow).

non-clinical colleagues to develop better diagnostic tests, drug therapies and vaccines and other preventive interventions.

#### PROTECTION OF RESEARCH PARTICIPANTS

Written consent was obtained from each patient presented in this article. He or she explicitly consented to the inclusion of background and clinical data, as well as radiographic images.

#### NON-CYSTIC FIBROSIS BRONCHIECTASIS (NCFB)

#### Case

A 67-year-old female lifelong non-smoker with a history of scoliosis and early osteoporosis developed a non-productive cough for 6 months, an unintentional weight loss, and daytime fatigue. She had experienced a seven-pound weight loss over 6 months, to a body mass index (BMI) of 17.5 kg/m2 (normal: 19–24 kg/m2 ). An avid swimmer and jogger, she had also noticed a gradual decline in her stamina, with an increase in shortness of breath with moderate exercise. Her primary care provider ordered a chest radiograph, which showed possible right middle lobe pneumonia. She was prescribed 10 days of levofloxacin, which did not improve her respiratory symptoms. She developed severe diarrhea, and on the seventh day of levofloxacin, her husband took her to the emergency department, where she was admitted for dehydration secondary to Clostridium difficile colitis. Because the cough had not improved with antibiotics, a CT of the chest was performed, showing right middle lobe and lingula bronchiectasis, nodularity, and mucus impaction (**Figure 1**). She was placed on airborne isolation for possible pulmonary tuberculosis. MTB PCR probe was negative on an TABLE 2 | Radiographic presentations of pulmonary NTM diseases.

fmicb-09-02901 November 29, 2018 Time: 13:6 # 3

induced sputum sample, so she was taken for bronchoscopy with lavage. These samples were smear positive for AFB, and 10 days later, grew M. avium. She was discharged from the hospital on a combination of ethambutol, rifampin, and azithromycin three times weekly, and after 3 months of treatment, her cough resolved and she began to gain weight. Repeat CT imaging at 6 months demonstrated fewer lung nodules, with less mucus impaction.

#### Discussion

The nodular-bronchiectatic phenotype of pulmonary NTM disease is also known as "Lady Windermere Syndrome," in reference to the character from Oscar Wilde's 1892 play entitled "Lady Windermere's Fan" (Reich and Johnson, 1992). While the term has had wide adoption in the medical literature, it is, in fact, a malapropism, since the character for which the syndrome is named is a young woman without respiratory symptoms of any kind. Further, the authors who originally named this syndrome believed that the infection resulted from women chronically suppressing their cough, leading to mucus inspissation in the longer airways of the right middle lobe and lingula. This hypothesis has more recently been challenged, since a hallmark of the nodular-bronchiectatic phenotype is chronic cough and sputum production (Olivier, 2016; Tsai et al., 2017). Reflex cough responses in elderly women with stable pulmonary NTM disease appear similar to age-matched controls, except for a mildly decreased urge-to-cough, arguing against cough suppression (Tsai et al., 2017).

Individuals with this phenotype are typically female, elderly, are tall with thin body habitus, have pectus excavatum, scoliosis, and a non-smoking history (Kim et al., 2008; Park et al., 2009; Lee et al., 2013). Radiographically, the right middle lobe and lingula are most often affected, though any area of the lung may be affected (**Figure 1**). Typically, the radiographic pattern is described as "tree-in-bud," indicating bronchiolar inflammation and mucus impaction. Coinfection with more than one species of Mycobacterium, or with Gram-negative bacteria, characterizes later, more advanced disease.

Symptomatically, patients with the nodular-bronchiectatic phenotype typically complain of chronic cough, which may or may not be productive, and may or may not be associated with hemoptysis, persistent fatigue, night sweats, and unintentional weight loss. The infection may be mistaken for active pulmonary tuberculosis, leading to airborne isolation and prolonged hospitalization, as happened with our patient. Treatment is typically a prolonged combination regimen of a rifamycin, most often rifampin, ethambutol, and a macrolide, typically azithromycin or clarithromycin. The frequency of dosing

of cystic bronchiectasis (yellow arrow).

depends on the extent of radiographic disease: mild nodularbronchiectatic disease may be treated with a thrice-weekly regimen, while more severe bronchiectasis requires a daily regimen. In the most severe cases, particularly those with cystic bronchiectasis and cavitary lung disease, a concurrent thriceweekly parenteral aminoglycoside, such as amikacin, may be necessary (Griffith et al., 2007). As with the M. abscessus group, surgical resection in localized or cavitary disease is an option where medical treatment has failed to eradicate infection. Since individuals with the nodular-bronchiectatic phenotype are typically elderly and frail, the risks of surgical resection may outweigh potential benefits, in which case a chronic suppressive strategy with antibiotics is a reasonable alternative.

Complete clearance of MAC infection is variable, and often depends on the severity of disease. Further limiting the successful clearance of MAC infection is the low rate of clinician adherence to the 2007 ATS/IDSA treatment guidelines (Adjemian et al., 2014a,b). With the guidelines-based treatment approach, more than 60% of patients with nodular-bronchiectatic disease will have treatment success (Wallace et al., 2014; Diel et al., 2018). These patients continue to be susceptible to reinfection, however, often with entirely new strains or subspecies. Overall, the recurrence of pulmonary MAC infection approaches 40%, with the highest rates of recurrence among the nodular-bronchiectatic cohort (Lee et al., 2015; Boyle et al., 2016). The rate of relapsed disease, where the same clinical isolate is again isolated within 12 months of supposed cure, is unknown. While environmental exposure has been proposed as a source of infection, it is unclear what role remediation and exposure avoidance have on preventing reinfection.

#### CYSTIC FIBROSIS LUNG DISEASE (CF)

#### Case

A 21-year-old man with CF presented to the outpatient clinic with worsening dyspnea, weight loss, fever, and hemoptysis over approximately 2 months. He had last been in clinic 3 months prior when he had reported feeling well. Induced sputum sample during that clinic visit grew scant mucoid fluoroquinoloneresistant Pseudomonas aeruginosa. Mycobacterial cultures were negative at that time, and his forced expiratory volume (FEV1: typically the best overall measure of lung function) was stable at 2.5 L (normal for patient's age: 4.5 L). His BMI was chronically low at 17.8 kg/m2 (normal: 19–24 kg/m2 ). At the current clinic visit, his FEV1 had declined to 2.1 L, and his sputum sample was a dark gray streaked with blood. Because his resting pulse oximetry reading was consistently in the upper-80% range, he was directly admitted to hospital, where sputum sample was smear positive for AFB. M. tuberculosis PCR probe was negative. Chest radiograph on admission demonstrated upper and midlung zone cystic bronchiectasis with a new possible left upper lobe infiltrate (**Figure 2**, left). CT of chest confirmed the development of a left upper lobe infiltrate, with new tree-in-bud attenuation (**Figure 2**, right). The sputum culture grew M. abscessus 5 days after admission.

#### Discussion

Patients with CF have a higher prevalence of NTM disease than the general population. Pathogenic NTM species are isolated from approximately 1 in 5 patients with CF, and the incidence appears to be increasing by 5% per year, depending on geographic location (Adjemian et al., 2014a, 2018). Among the NTM species affecting this patient population, approximately 60% of isolates are MAC, while almost 40% are MABSC. MABSC is comprised of three subspecies: M. abscessus, M. bolletii, and M. massiliense, all of which are rapidly growing organisms. MABSC is a notoriously difficult infection to treat, owing in part to the erm(41) gene, which confers inducible macrolide resistance (Adjemian et al., 2018). The subspecies M. massiliense is an exception, however, since it lacks a functional erm(41) gene, and is therefore more easily eradicated (Kim et al., 2010; Roux et al., 2015). Patients with CF who develop MABSC infection typically have a lower BMI and, paradoxically, a higher FEV1 than those without MABSC infection (Adjemian et al., 2018).

Infection with MABSC is considered a relative contraindication to lung transplantation, since MABSC infection may prove fatal in the post-transplant period, when recipients are on maximal immunosuppressive therapy (Chalermskulrat et al., 2006; Gilljam et al., 2010). Because subjects with CF have progressive lung disease, lung transplantation is often a consideration at the later stages of disease. Our patient was concurrently followed by the lung transplant clinic, though he had never been listed. His sputum isolate was ultimately identified as subspecies M. massiliense, and he was successfully treated with a regimen consisting of azithromycin, amikacin, and cefoxitin. Sputum samples at 3, 6, and 9 months were negative for NTM species. His hemoptysis resolved, and he did not require supplemental oxygen at his three-month follow-up appointment.

#### CHRONIC OBSTRUCTIVE LUNG DISEASE (COPD)

#### Case

A 55-year-old man with a history of COPD associated with smoking developed a chronic cough productive of sputum, malaise, and night sweats. The social history was remarkable

FIGURE 3 | Left: chest radiograph demonstrating a right upper lobe cavitary lesion on a background of emphysema (green arrow). Right: axial CT image demonstrating the same cavitary lesion in the right lung (yellow arrow). Reprinted with permission of the American Thoracic Society. Copyright© 2018 American Thoracic Society. Fennelly et al. (2016) is an official journal of the American Thoracic Society.

the inner wall of the lung cavity, with many AFB seen at higher power using Ziehl Nissen stain (right, yellow arrows). Reprinted with permission of the American Thoracic Society. Copyright© 2018 American Thoracic Society. Fennelly et al. (2016) is an official journal of the American Thoracic Society.

for use of a hot tub at his home. Sputum microbiology was notable for 3+ AFB on smear and three cultures grew M. abscessus, susceptible to amikacin and linezolid, and intermediate to clarithromycin (without erm gene testing) and cefoxitin. Pulmonary function testing showed airflow obstruction and a low diffusing capacity, and imaging demonstrated a right upper lobe cavity and emphysematous changes, consistent with COPD (**Figure 3**). He and his wife had just retired and sold their home in northern Florida. They were en route to vacation in the American West in their recreational vehicle (RV), hoping to receive some oral antibiotics and be on their way. His symptoms responded well to treatment with intravenous amikacin once daily and intravenous imipenem-cilastatin twice daily and azithromycin (home health nursing visited him in a state forest campground where they lived in their RV during his treatment). However, 4 months after treatment he was admitted to the hospital with new shortness of breath, fevers, and with ground-glass opacities, consistent with parenchymal inflammation, in the posterior aspects of both upper lobes. Bronchoalveolar lavage (BAL) grew only M. abscessus, and the BAL cell count was markedly lymphocytic. This pattern was consistent with an allergic alveolitis due to a spill of the cavity, so

he was treated with oral corticosteroids and his antimycobacterial regimen intensified by the addition of linezolid once daily. As the cavity was the major site of disease and his lung function was only mildly decreased, he was referred for surgical resection of the right upper lobe. The histopathology of the cavity was remarkable for aggregates of AFB along the inner wall of the cavity (**Figure 4**). M. abscessus grew from a culture of the resected lung cavity within 3 days. Scanning electron microscopy of the inner wall of the resected cavity demonstrated bacilli embedded within a matrix. There were 3.83 × 107 colony forming units (CFU) of total bacteria in a 0.5 g sample from the lung cavity; 7.17 × 105 CFU were in biofilm. The bacterial composition was 97.7% mycobacterial using 16s ribosomal RNA sequencing (Fennelly et al., 2016). The patient's symptoms and imaging improved post-operatively, and all sputa specimens were negative on culture in the subsequent 10 months of his tour of the West.

#### Discussion

Patients with COPD often have emphysema that is typically more prominent in the upper lobes. The 'blebs' or holes within the lung parenchyma are presumably infected by NTM. The mode of infection is not well understood, but we suspect that it was likely due to inhalation in this case. We see a similar pattern of Aspergillus fumigatus infecting the upper lobes of COPD patients, and Aspergillus in the most ubiquitous airborne mold. As TB often presents with upper lobe cavities, COPD patients with AFB in their sputum are often initially placed in respiratory isolation as TB suspects unless a rapidly done molecular diagnostic can confirm that the AFB are not M. tuberculosis.

Pulmonary NTM infections in COPD patients have a low cure rate, generally considered to be about 50% compared to about 75% in patients with idiopathic bronchiectasis (Jones et al., 2018). Whether or not this lower cure rate is due to the presence of cavities is unknown. Surgical resection of a cavity can be curative, as in this case. Unfortunately, many patients with cavitary disease have lung function that it so diminished that they cannot tolerate surgery. Surgical resection may also be contraindicated if there is extensive bilateral disease. lower lobes.

fmicb-09-02901 November 29, 2018 Time: 13:6 # 6

However, medical treatment can have a role in preventing further progression of disease, possibly by suppressing bacterial growth within cavities or by protecting healthy lung tissue by eradicating small amounts of bacteria that are spread from diseased areas. **Figure 5** shows the chest radiographs of a woman with COPD who had cavitary pulmonary MAC disease (**Figure 5**, Left). Although her sputum remained AFB smear- and culture-positive during her course of treatment, her symptoms had improved and there was no further radiographic evidence of disease progression. Unfortunately, she stopped all her medications when she was given a diagnosis cervical cancer and became depressed, and she was lost to follow-up. Approximately 6 months later she was admitted to the intensive care unit, and a repeat chest radiograph showed dramatic progression of disease (**Figure 5**, Right). The right upper lobe cavity appeared to have extended and spilled, destroying her entire right lung and significantly involving the left. She died 2 weeks later. COPD appears to be the most important underlying condition leading to mortality in patients with pulmonary NTM infections (Hayashi et al., 2012; Ito et al., 2012; Mirsaeidi et al., 2014; Gommans et al., 2015; Jones et al., 2018), although fibrocavitary disease without COPD is also associated with increased mortality (Fleshner et al., 2016).

### PRIMARY CILIARY DYSKINESIA (PCD)

#### Case

A 53 year old woman is followed for bronchiectasis, sinusitis and chronic lung infections with MAC and P. aeruginosa. She had no respiratory distress as an infant, but she began to develop recurrent sinus infection and a productive cough around age four. At the age of 18 she was diagnosed with bronchiectasis. She had been evaluated for CF with sweat chloride tests were negative on three occasions. She has two children, and no other family members have respiratory disease. On exam now, she is thin with a body-mass index of 17.9 (normal: 19–24 kg/m2 ). On lung exam she has scattered wheezes and rales in the bases. She inhales nebulized hypertonic saline twice daily and engages in aerobic exercise daily for airway clearance.

Imaging of her sinuses shows chronic sinusitis, with airfluid levels in both maxillary sinuses and opacified ethmoid and frontal sinuses, in spite of evidence of prior surgeries. Computed tomography (CT) of the chest is remarkable for severe bronchiectasis, predominantly in both lower lobes (**Figure 6**).

At the age of 37, she was first diagnosed with pulmonary MAC infection after presenting with worsening cough. She improved on treatment with clarithromycin, ethambutol and rifampin for 9 months. However, her cough returned several months after treatment was stopped, and she was restarted on the same treatment except with azithromycin instead of clarithromycin. Unfortunately, she noticed difficulties discriminating red and green colors and was diagnosed with optic neuritis secondary to ethambutol but this resolved after discontinuation of the drug. She was treated with inhaled amikacin but it was stopped due to severe hoarseness. Linezolid was recently added but the discontinued after she developed numbness and tingling in her fingers and toes consistent with peripheral neuropathy.

At the age of 47, the diagnosis of PCD was confirmed by low nasal nitric oxide of 15 nL/min and genetic analysis demonstrating two disease-causing mutations in DNAH11. Electron microscopy showed no structural changes of the cilia as seen in classic PCD.

P. aeruginosa was first cultured from her sputum in early 2017, but she was feeling relatively well. She received three courses of oral ciprofloxacin without eradication. In the fall of 2017, she complained of more cough and sputum production. She was treated with cefepime and tobramycin by the intravenous route for 2 weeks to treat a bronchiectasis exacerbation due to P. aeruginosa. She had baseline mild tinnitus that did not change. A follow-up audiological evaluation 1 month later showed the development of mild to moderate high frequency sensorineural hearing loss bilaterally, although the patient did not perceive hearing loss. Follow-up audiological evaluations after another month and then 6 months later showed no improvement in the hearing acuity, but it remained stable. Unfortunately, her Pseudomonas infection now appears chronic. She is feeling relatively well on suppressive therapy with inhaled aztreonam.

#### Discussion

PCD, or immotile cilia syndrome, is an autosomal recessive disorder of motile cilia dysfunction resulting in impairment of mucociliary clearance. It is heterogenous, with over 30 genetic variants described to date and a wide range of clinical manifestations. Unlike the patient above, approximately 80% of newborns have neonatal respiratory distress. About one-half of PCD patients have 'laterality defects' such as situs inversus, in which the organs are on the opposite side of the body due to defects in the embryonic nodal cilia. In children, CF and PCD may both present with respiratory infections and distress. In CF, the problem is due to alterations of the airway surface fluid,

whereas in PCD the problem is with poor movement of the fluid due to the dysfunctional cilia. Both result in bronchiectasis (dilated, chronically inflamed bronchi), but the location tends to be in the upper lobes in CF and in the lower lobes in PCD. Nearly all PCD patients have rhinosinusitis. Infertility is a possibility, but not universal as in this case. The median age of diagnosis in childhood is about 5 years of age, but the age of diagnosis in adults is widely variable. Diagnosis has traditionally been difficult due to variable availability and accuracy of tests such as transmission emission microscopy to evaluate 'classic' ciliary ultrastructural defects and nasal nitric oxide measurements. However, the increased availability of genetic testing has been a significant advance. A recent clinical practice guideline provides a very helpful diagnostic algorithm (Shapiro et al., 2018).

Most PCD patients live an active life and have a normal lifespan, although lung infections can interfere with their normal social functions. The rate of decline in their lung function is slower than in CF. The rates of NTM infections among patients with PCD is not known. In a recent registry study, PCD patients were less likely to have NTM infections than those with bronchiectasis associated with gastroesophageal reflux disease (GERD).

This case highlights problems of drug toxicity experienced by most patients with NTM lung diseases, who are also frequently co-infected with Gram-negative bacteria or fungal pathogens. In review, this patient has had adverse reactions to ethambutol (optic neuritis), inhaled amikacin (laryngitis), linezolid (peripheral neuropathy), and intravenous tobramycin (ototoxicity). Due to the multiple adverse reactions, her chronic MAC infection now can be treated only with azithromycin and rifampin, a suboptimal regimen for cure, which is probably unlikely to occur due to the severity of her bronchiectasis. The goal of therapy for both the MAC and the Pseudomonas is now suppression of bacterial growth to prevent exacerbations and further lung destruction. Such adverse reactions to drugs limit the armamentarium available to physicians to treat patients, and contribute to the urgent need for additional antibacterial drugs.

#### HYPERSENSITIVITY PNEUMONITIS (HP)

#### Case

One of us (KF) was consulted on the case of a 52 year-old pulp mill worker for evaluation of possible underlying occupational lung disease. He had been referred to the infectious disease service after a surgical lung biopsy at an outside hospital showed non-caseating granulomas and AFB. The lung tissue culture had grown MAC. The patient had noted progressive shortness of breath. On physical examination, the patient had a very rapid breathing rate in spite of being on supplemental oxygen by nasal cannula with a reservoir device at 15 l/min. There were inspiratory crackles on lung exam. The lung imaging was remarkable for diffuse 'mosaic attenuation' and fine nodules (**Figure 7**) which was not consistent with what is usually seen in MAC infections. It was originally suspected that he might have HP due to airborne molds within the pulp mill. However, the history revealed that he had not worked inside the pulp mill for several years due to a severe back injury. He worked in a control room in another building at a distance from the plant. To manage his back pain, the patient had the habit of soaking in a hot tub for 30 min in the morning after awakening and then again in the evening when he returned from work. The hot tub was in a room just outside his bedroom. He was too short of breath to be able to do pulmonary function testing. We considered performing

a bronchoscopy to obtain BAL fluid for cell counts, but we realized that both HP and mycobacterial infection would be predominantly lymphocytic. Given his tenuous respiratory status and his tissue diagnosis, the risk of the procedure far outweighed any benefit. He was treated with both systemic corticosteroids for the HP and with azithromycin, rifampin and ethambutol for the MAC infection. He began to improve within a few days in the hospital, and was discharged home with the advice to drain the hot tub after obtaining a water sample for culture. He sent the water to the local state public health laboratory and received a report that it grew MAC. Unfortunately, when we called to request the isolate to determine if it was identical to the clinical one, the laboratory in a southeastern state had already discarded it because they were overwhelmed with such cultures. When we saw him 18 months after his removal from exposure and initiation of treatment, his symptoms, exam, imaging and pulmonary function tests were completely normal.

#### Discussion

This was a classic case of 'hot tub lung' due to inhalation of NTM. This form of HP was first described in case reports in 1997, and multiple cases have now been reported in the literature. In a retrospective case series of 21 patients at a referral center, all patients presented with shortness of breath and cough and about half were hypoxemic. MAC was isolated from both the hot tub water and the respiratory secretions or tissue in all cases (Hanak et al., 2006). All improved with avoidance of exposure, and there was a mix of additional corticosteroid and antimycobacterial therapy, but five (24%) received neither. Thus, avoidance of exposure appears to be the most important element. However, in patients with severe illness like our case, more aggressive treatment is warranted. Some of the more interesting case reports have involved multiple immunocompetent family members developing HP due to MAC and possibly other NTM (Mangione et al., 2001; Kitahara et al., 2016).

An occupational respiratory illness associated with NTM that emerged in the last decade is HP associated with metalworking fluids in both the United States and the United Kingdom (Dawkins et al., 2006). These fluids were found to be contaminated with a rapidly growing Mycobacterium now known as M. immunogenum (Wallace et al., 2002).

Mycobacterium chelonae was isolated from a bassoon played by a professional musician diagnosed with HP, but molds were also isolated and no NTM or molds were isolated from the BAL from the patient (Moller et al., 2017). Thus, the association may not be causal. NTM contamination in a water-damaged building was recently associated with asthma symptoms (Park et al., 2017). Environmental sampling for NTM is not commonly done in indoor environments, suggesting a need for further research about environmental exposures to airborne NTM.

#### DISSEMINATED DISEASE

#### Case

A 49-year-old denied any infections or hospitalizations in his childhood. As a young adult he had histoplasmosis lymphadenitis and was treated successfully. He did well until 47 years of age when he presented to a hospital complaining of diarrhea after having several year history of GERD, intermittent nausea and emesis. Colonoscopy was performed and pathology showed Periodic Acid Schiff stain (PAS) positivity, so empiric treatment for Whipple's disease, a rare systemic infection caused by the bacterium Tropheryma whipplei, was initiated. One month later, he presented to a large tertiary hospital with mycobacteremia, hypotension and sepsis. He was diagnosed with idiopathic CD4 lymphopenia and disseminated MAC. He suffered multiple cerebral embolic events, had a tracheostomy placed, underwent PEG placement and suffered extensive end-organ damage from long term aminoglycoside therapy. His GI disease was indicative of his overall burden of disease. He was referred to the NIH after the diagnoses of two variants (IL12Rβ1 c.512 A > C, p.Q171P; and IL12Rβ1 c. 1442A > G, p.Y481C) were found in IL-12 receptor 1. He required years of intensive multidrug therapy in addition to immune therapies including exogenous interferon gamma.

#### Discussion

Genetic susceptibility to disseminated mycobacterial infection is well described and should be considered in anyone who presents with disseminated disease without HIV infection (Wu and Holland, 2015). Even diseases that are often diagnosed in childhood can present with disseminated infection in adulthood when their mutation results in a less severe phenotype (Hsu et al., 2018). Host control depends on the interleukin 12-interferon pathway that connects myeloid and lymphoid cells. The genetic susceptibilities, if inherited, can be elucidated by a careful family medical history. Autosomal dominant patterns of inheritance include: IFNR1/R2, STAT1, IRF8, GATA2, IL12Rβ1/R2 X-linked: NEMO. Autosomal recessive patterns of inheritance typically present early in life and would not have been considered in this age patient. Non-HIV Acquired immunodeficiency such as autoantibodies to interferon gamma (IFN) would have been another consideration in this patient and one that is important to consider in adults with disseminated infection. However, in the United States, women have been identified more frequently than men, and have mainly been described in the East Asian population.

IL-12Rβ1 encodes the 1 chain of IL-12 and IL-23 receptors and activation of pSTAT4 and is important in the subsequent signaling of IFN and activation of macrophage intracellular killing. IL-12Rβ1 has been reported in more than 200 patients worldwide (de Beaucoudrey et al., 2010). Typically, they will present after BCG vaccination in countries where TB is endemic. However, in the United States, these patients may present later in life, as our patient presented. This case of a primary immune deficiency that phenotypically presented in adulthood highlights the importance of evaluating genetic disorders that may predispose individuals to disseminated infection with a low virulent organism in the absence of known risk factors.

#### Case

A 32-year-old Filipina woman presented to her primary care provider with fevers, neck and back pain, and cervical

lymphadenopathy. She was diagnosed with communityacquired pneumonia, and treated with azithromycin and oral corticosteroids as well as a subsequent course of doxycycline with more oral corticosteroids. While her pain improved on the corticosteroids, she started having night sweats off of prednisone. An eventual CT of her neck showed destruction of her C3 vertebra, and lymphadenopathy. A PET/CT showed uptake in activity in lymph nodes, femurs, left acetabulum, left pubic ramus, sacrum, right ischium, both iliac bones, ribs, and clavicles. She underwent C3 corpectomy with fusion; followed by right femoral curettage 1 month later. When AFB+ organisms were seen, an interferon gamma release assay (IGRA) was indeterminate. Cultures grew MAC. Three months after the first surgery, she continued to have pain and underwent debridement of an epidural abscess in T8, involving a laminectomy, foraminectomy and hardware placement of screws at T6, T7, T10, and T11. Her disease continued to worsen despite optimal multi-drug therapy. Subsequent testing at the National Institutes of Health (NIH) showed very high titer autoantibodies to Interferon gamma. She was referred to the NIH and continued on multi-drug antibiotic therapy and given rituximab with improvement in her disease. Unfortunately, when her B-cells returned after 2 years, her disease recurred and she remains on therapy for her infection and is getting repeat rituximab infusions for the IFN autoantibodies.

#### Discussion

Autoantibodies to IFN were first reported by Doffinger et al. (2004). An adult-onset immunodeficiency due to these autoantibodies in Thailand and Taiwan was described in 2012 in a population of HIV negative patients presenting with opportunistic infections (Browne et al., 2012). They have since been described in patients with thymic neoplasia (Burbelo et al., 2010). An indeterminate IGRA test is characteristic in these patients, as sera from them will block positive controls for this commercial test and will therefore be reported as indeterminate. In vitro, the pSTAT1 activation is negative when patient sera is tested in the presence of normal PBMCs. Especially in Asians, one should consider autoantibodies to IFN when encountering an adult with disseminated NTM, particularly when there is no family history of disease.

#### VERTEBRAL OSTEOMYELITIS

#### Case

A 16-year-old girl was evaluated for progressive mid-thoracic back pain. She reported the onset after abatement of symptoms of a flu-like illness with fever, nasal congestion, sore throat and non-productive cough and muscle aches. She was a competitive roller-skater, and she had experienced multiple falls in the prior year. She recalled multiple ecchymoses and abrasions due to these falls. She had no history of unusual infections earlier in her life, and there was no suggestion of immunodeficiency in her laboratory data. The C-reactive protein (a non-specific marker of systemic inflammation) was

minimally elevated at 1.3 mg/dL. Her examination was normal with no neurological deficits. A CT scan of her lungs was normal. A radionuclide bone scan demonstrated increased uptake in the vertebral body of T9, and magnetic resonance imaging (MRI) showed a soft tissue mass anterior to the vertebral bodies of T8, T9, and T10. Thoracoscopic biopsy of the T9 vertebral body demonstrated necrotizing granulomas of the bone; special stains for bacteria, mycobacteria and fungi were negative (**Figure 8**). She was treated initially for both M. tuberculosis and NTM with isoniazid, rifampin, ethambutol and clarithromycin. After the culture grew only M. abscessus, she was treated with only clarithromycin; the other drugs for tuberculosis were discontinued. A follow-up MRI showed resolution of the edema and overall improvement at 4 months. The clarithromycin was discontinued after 6 months, and she was well with a normal C-reactive protein 3 months later. She was advised to discontinue roller-skating and other potentially traumatic activities and to be closely monitored for relapse.

#### Discussion

Although most cases of disseminated NTM disease suggest the presence of an immunodeficiency, NTM infections can occasionally occur at distant sites from the lung in special situations. Obviously these organisms can gain access to bone or other internal structures during penetrating trauma. However, this case was associated with blunt trauma and so illustrates the principle of 'locus minoris resistentiae,' i.e., a place of less resistance, in this case to microbes. This unusual case was previously published along with two similar cases in 2001 (Chan et al., 2001). In retrospect, such a case now would probably be treated not only with a macrolide like clarithromycin, but also with at least one intravenous medication such as amikacin. The isolate in this case was most likely a M. massiliense subspecies that was susceptible to clarithromycin. Interestingly, shorter courses of therapy than are used for pulmonary disease are acceptable for bone infections.

### NOSOCOMIAL INFECTIONS WITH Mycobacterium chimaera AFTER CARDIAC SURGERY

fmicb-09-02901 November 29, 2018 Time: 13:6 # 10

#### Case

A 64-year-old man had cardiac surgery for a mitral valve porcine mitral valve replacement and a new prosthetic aortic valve in March of 2012. His postoperative course was uneventful until 5 months after the surgery when he developed persistent fevers between 103 and 104 degrees Fahrenheit. Blood cultures were negative, and transthoracic and transesophageal echocardiograms were normal. He was treated empirically with both oral and intravenous antibiotics for culture negative endocarditis in the setting of his recent surgery. In December 2012 (9 months after surgery) blood cultures drawn 30 days prior grew M. chimaera (a species in the M. avium complex). Despite optimal medical management with intravenous and oral multi-drug therapy, he remained persistently bacteremic for 2 years. Unfortunately, despite multi-drug therapy and close medical follow-up, the patient died of his persistent bacteremia.

#### Discussion

A prolonged outbreak of M. chimaera infections after openchest cardiac surgery was first reported by Swiss investigators in 2015. After the recognition of a cluster of cases of disseminated M. chimaera after cardiac surgery at the same hospital as our patient's, the US Centers for Disease Control and Prevention (CDC) and the Pennsylvania Department of Health investigated the outbreak. They found that the heatercooler devices (HCDs) used to maintain the patients' body temperature during extracorporeal circulation (also known as cardiac bypass) were contaminated with M. chimaera, confirming similar findings by the Swiss investigators (Perkins et al., 2016). The isolates obtained from HCDs from three different European countries were nearly identical to those from the manufacturing site in another study. The results of these investigations strongly suggested a point-source contamination in the manufacturing of a specific HCD. There is a growing body of literature on this problem, and investigations are on-going.

Whole exome sequencing revealed that our patient had a mutation of interferon regulatory factor 8 (IRF8), which regulates expression of interferon-alpha and interferon beta genes. IRF8 is critically important in immune responses to intracellular pathogens, especially mycobacteria, involving the production of interleukin-12 in response to interferon-gamma (Hambleton et al., 2011). This mutation likely contributed to his susceptibility and death due to this infection.

### CONCLUSION

The cases presented in this paper demonstrate that NTM infections vary widely in presentation, treatment, and clinical outcome. While ubiquitous in the environment, NTM species tend to infect certain vulnerable patient populations, and these infections can often be organized into clinical phenotypes. While many NTM species have low pathogenicity, some opportunistic pathogens are important causes of human disease. Infection with these organisms, including MAC, MABSC, M. kansasii, M. xenopi, and others, are increasing in incidence and geographic distribution. Most human disease is pulmonary, primarily affecting immunocompetent patients with underlying structural lung disease such as bronchiectasis or COPD. Disseminated disease is usually more life-threatening, and primarily occurs in immunocompromised individuals. Among those most severely affected by disseminated disease are children with specific primary immunodeficiencies, and those with acquired T cell immunodeficiency due to anti-cytokine antibodies or iatrogenic administration of immunosuppressive medications, particularly tumor-necrosis factor alpha antagonists.

We hope that the host variability in NTM disease presented in this paper serves to stimulate and inspire basic and translational scientists to consider a similar variety of innovative interventions. For example, the poor response to treatment and increased mortality seen in fibrocavitary lung disease might be improved by drugs targeting biofilm dispersion (COPD Case). Inhaled liposomal amikacin was recently approved for pulmonary MAC disease in the United States, and translational studies on its role in biofilm infections may be a next step. Elucidation of the pathogenesis of non-CF bronchiectasis in tall, thin women may enable us to 1 day prevent the development of the disease. Candidate areas of research for the nodular-bronchiectasis MAC phenotype include leptin and ghrelin hormone influences on the immune response, cellular senescence, and the aging lung. In CF, research is needed on potential airborne transmission of M. abscessus, biofilm formation in the airways by M. abscessus, the effect of new CFTR modulator drugs on mycobacterial infection, among others. In PCD, it is possible that drugs improving the function of cilia may have a role in preventing NTM infections, and perhaps gene mutations could be corrected in the future. HP could be prevented by improved water disinfection techniques, and disseminated disease acquired during cardiac bypass could also be prevented by improved disinfection of heart-lung machines. Disseminated diseases associated with immunodeficiencies could be prevented by better immunomodulatory drugs that could correct such defects. Obviously, most patients would benefit from simpler, shorter and more effective drug treatment regimens against the mycobacteria, and the role of inhaled liposomal amikacin in such regimens continues to be defined.

The cases that we have presented illustrate the broad spectrum of NTM disease. We hope that these clinical vignettes have provided non-clinical scientists with a better understanding of human NTM diseases.

### AUTHOR CONTRIBUTIONS

All authors contributed sections to the manuscript, and all reviewed and contributed to revisions.

### REFERENCES

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diffuse panbronchiolitis. J. Korean Med. Sci. 24, 427–432. doi: 10.3346/jkms. 2<IP_ADDRESS>7

mycobacterial lung infections. J. Appl. Physiol. 122, 1262–1266. doi: 10.1152/ japplphysiol.00939.2016

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Swenson, Zerbe and Fennelly. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Assessing Response to Therapy for Nontuberculous Mycobacterial Lung Disease: Quo Vadis?

Christopher Vinnard1 \*, Alyssa Mezochow2 , Hannah Oakland3 , Ross Klingsberg3 , John Hansen-Flaschen2 and Keith Hamilton2

1 Public Health Research Institute, New Jersey Medical School, Newark, NJ, United States, 2 Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States, 3 Department of Medicine, Tulane University School of Medicine, New Orleans, LA, United States

Assessing progression of disease or response to treatment remains a major challenge in the clinical management of nontuberculous mycobacterial (NTM) infections of the lungs. Serial assessments of validated measures of treatment response address whether the current therapeutic approach is on track toward clinical cure, which remains a fundamental question for clinicians and patients during the course of NTM disease treatment. The 2015 NTM Research Consortium Workshop, which included a patient advisory panel, identified treatment response biomarkers as a priority area for investigation. Limited progress in addressing this challenge also hampers drug development efforts. The Biomarker Qualification Program at the FDA supports the use of a validated treatment response biomarker across multiple drug development programs. Current approaches in clinical practice include microbiologic and radiographic monitoring, along with symptomatic and quality-of-life assessments. Blood-based monitoring, including assessments of humoral and cell-mediated NTMdriven immune responses, remain under investigation. Alignment of data collection schemes in prospective multicenter studies, including the support of biosample repositories, will support identification of treatment response biomarkers under standard-of-care and investigational therapeutic strategies. In this review, we outline the role of treatment monitoring biomarkers in both clinical practice and drug development frameworks.

Keywords: nontuberculous mycobactena, biomarker (development), response, therapeutics, clinical trial, radiography, quantitative culture

Nontuberculous mycobacteria (NTM) are ubiquitous environmental organisms capable of causing significant morbidity (Yeung et al., 2016) and mortality (Vinnard et al., 2016; Diel et al., 2018), predominantly in the form of chronic lung disease (Wassilew et al., 2016). Profound knowledge gaps regarding the diagnosis of NTM lung infections (Griffith et al., 2007) include distinguishing NTM colonization from infection (van Ingen, 2015), identification of patients most likely to benefit from treatment (Mirsaeidi et al., 2014), selection of an optimal initial drug regimen (Philley and Griffith, 2015), and classification of treatment endpoints. As an explanation for these knowledge gaps, the formal study of NTM lung disease is relatively recent compared to tuberculosis, and basic understanding of NTM lung disease pathophysiology has only recently emerged.

In this review, we address uncertainties regarding the clinical "waypoints" of NTM treatment. Intermediate and definitive markers of treatment response provide crucial feedback to NTM patients, their clinicians, and developers of novel therapies. We outline the role of

#### Edited by:

Rustam Aminov, University of Aberdeen, United Kingdom

#### Reviewed by:

David E. Griffith, The University of Texas Health Science Center at Tyler, United States Debra Hanna, Bill & Melinda Gates Foundation, United States

\*Correspondence:

Christopher Vinnard<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

Received: 02 July 2018 Accepted: 01 November 2018 Published: 20 November 2018

#### Citation:

Vinnard C, Mezochow A, Oakland H, Klingsberg R, Hansen-Flaschen J and Hamilton K (2018) Assessing Response to Therapy for Nontuberculous Mycobacterial Lung Disease: Quo Vadis?. Front. Microbiol. 9:2813. doi: 10.3389/fmicb.2018.02813

**20**

treatment monitoring biomarkers in both clinical practice and drug development. We summarize the existing knowledge base regarding biomarkers of treatment response for NTM disease and we identify areas for ongoing and potential future investigation.

### THE UTILITY OF A TREATMENT RESPONSE BIOMARKERS IN CLINICAL CARE AND DRUG DEVELOPMENT

In his seminal framework for defining surrogate endpoints in clinical trials, Ross Prentice proposed that a valid surrogate endpoint must "capture" the underlying relationship between the intervention and the clinical endpoint, allowing for comparison of intervention groups based on the surrogate rather than the definitive clinical endpoint at any time during the followup period (Prentice, 1989). By comparison, the concept of a treatment monitoring biomarker is less restrictive than a surrogate endpoint. The Institute of Medicine defines a biomarker as "a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or biologic responses to a therapeutic intervention" (Institute of Medicine (US) Committee on Qualification of Biomarkers and Surrogate Endpoints in Chronic Disease, 2010).

The BEST (Biomarkers, EndpointS, and other Tools) Resource, published by the FDA-NIH Biomarker Working Group, provides descriptions and examples of disease monitoring biomarkers, which are "assessed serially over time to measure presence, status, or extent of disease or medical condition, or to provide evidence of an intervention effect or exposure, including exposure to a medical product or environmental agent" (Cagney et al., 2017).

Thus, the interpretation of a monitoring biomarker is not based on a snapshot level, as with a baseline clinical predictor of response to therapy, but rather on changes in values during serial measurements. As examples, quantification of HIV RNA in blood serves as a monitoring biomarker for the clinical efficacy of antiretroviral therapy, and the amount of air that an individual can forcefully exhale in one second (FEV1) reflects response to chronic obstructive pulmonary disease management. Ideally, a treatment monitoring biomarker could be readily adopted into existing procedures that support routine use in clinical settings (Nahid et al., 2011).

The therapeutic approach to NTM lung disease lacks the evidence base of tuberculosis, where standard-of-care combination drug regimens are based on randomized clinical trials that enrolled tens of thousands of tuberculosis patients. Unlike tuberculosis, the NTM lung disease research agenda has not been propelled by public health pressure or urgency. Consequently, much of the clinical approach to NTM disease rests on a foundation of observational data and expert opinion, supported by only a few randomized clinical trials (Research Committee of the British Thoracic Society, 2001). In general, this evidence base has demonstrated that antimycobacterial therapies are less effective for NTM lung disease than tuberculosis (Griffith et al., 2007). The 2015 NTM Research Consortium Workshop, which included a patient advisory panel, identified treatment response biomarkers as a priority area for investigation (Henkle et al., 2016). One or more treatment response biomarkers would help to answer a fundamental question for clinicians and patients during the course of NTM disease treatment: is the current therapeutic approach on track toward a clinical cure?

This question has a particular significance for NTM disease given the complexity of treatment and the frequency of drugrelated adverse events. Interval monitoring of treatment response would support clinical decision-making with regards to the intensification or de-escalation of therapies. When an initial period of intravenous therapy is used, for example with disease caused by M. abscessus complex, the optimal time point for transitioning to an all-oral regimen is uncertain. Surgical interventions may be the most appropriate approach for a subset of patients with severe M. abscessus complex lung disease, but potentially could be deferred in a subset of patients that are responsive to medical management (Jarand et al., 2011). Such decision nodes provide a potential point of application for a treatment monitoring biomarker. Monitoring biomarker response early in treatment could also inform clinical decisionmaking in advance of drug susceptibility testing (DST) results, which have a delay of several weeks and lack correlation between in vitro susceptibility and clinical outcomes for many antibiotics.

Aside from their value in clinical management decisions, treatment monitoring biomarkers support drug development efforts. According to guidelines established by the Food and Drug Administration (FDA), the use of a biomarker in the context of drug development relies on "qualification" that specifies its contextual use and interpretation. The Biomarker Qualification Program at the FDA provides a pathway for a particular biomarker to become accepted into the regulatory framework independent of approval of a specific drug (Amur et al., 2015). This program was recently updated in response to the 21st Century Cures Act, establishing formal qualification plans for biomarkers and other drug development tools. As an example, the Critical Path Institute (Tuscon, AZ, United States) is evaluating sputum levels of lipoarabinomannan, a component of the mycobacterial cell wall, as a non-culture based measure of tuberculosis treatment response, which could be incorporated into adaptive clinical trial designs (FDA, 2018a). Similarly, a qualified biomarker of treatment response for NTM disease would support drug development efforts across multiple programs, a notable advantage given the likelihood that combination antimicrobial therapies will remain essential to future drug regimens.

#### CURRENT APPROACHES TO MONITORING TREATMENT RESPONSE TO NTM DISEASE

#### Microbiologic Monitoring

Presently, culture of serial sputum samples serves as the primary biomarker for monitoring the response to treatment

of mycobacterial lung infections. In 2016, an expert panel composed of NTM clinicians reached consensus definitions for several endpoints related to the treatment of NTM lung disease, including cure, relapse, and re-infection (van Ingen et al., 2018). Serial culturing of sputum and deep respiratory samples during treatment of NTM lung disease provides the basis for definition of microbiologic cure, which is defined as 3 negative sputum cultures collected at least 4 weeks apart.

Interval microbiologic data may also provide insight into the effectiveness of therapy when the microbiologic cure endpoint has not yet been reached. Mycobacterial cultures from respiratory specimens can be classified semiquantitatively according to growth characteristics in liquid and solid media (Griffith et al., 2015; **Table 1**). Using this scoring system, Griffith et al. (2015) analyzed semiquantitative sputum culture scores on 180 patients with lung disease caused by M. avium complex (MAC), grouping patients into converters and nonconverters based on sputum culture status after 12 months of treatment. Although baseline semi quantitative culture scores were not significantly different between converters and nonconverters, the change in semi quantitative culture scores after 2 months of MAC-directed treatment was highly predictive of sputum culture conversion, with each 1-point decline in the scoring scale associated with a 20% increase in the likelihood of achieving conversion at 12 months. Importantly, early microbiologic response also correlated with symptomatic and radiographic improvements, suggesting that semi quantitative culture data can be explored as a surrogate endpoint in drug development. One limitation of this approach may be that some NTM lung disease patients do not expectorate sputum (Huang et al., 1999), although proper application of sputum induction technique considerably improves microbiologic yield (Guiot et al., 2017).

Interval treatment response monitoring based on microbiologic data is supported by recently published NTM lung disease treatment guidelines. The British Thoracic Society recommends obtaining interval sputum cultures every 4– 12 weeks during treatment to assess microbiologic response (Haworth et al., 2017). Similarly, consensus recommendations provided by the US Cystic Fibrosis Foundation and the European Cystic Fibrosis Society for the management of NTM lung disease include interval monitoring of sputum culture every 4–8 weeks during treatment (Floto et al., 2016). An update from the American Thoracic Society/Infectious Disease Society of America regarding management of NTM lung disease is currently in preparation. Unlike tuberculosis, there are no data supporting non-culture based microbiologic monitoring during NTM lung disease treatment.

### Radiographic Monitoring

Radiographic imaging studies have long served an essential role, not only in diagnosing NTM lung infection and determining the extent of disease, but also in assessing response to treatment (Griffith et al., 2007). A simple chest radiograph scoring system with 3 tiers (improved, no change, or worsened in comparison with the preceding examination) was predictive of sputum culture conversion among MAC lung disease patients,

with repeated routine chest radiography performed a mean of 59 days after the baseline (pre-treatment) examination (Griffith et al., 2015). Computed tomographic (CT) imaging considerably improves assessment and comparison of NTM disease extent compared to chest radiographs. Indeed, several scoring criteria for CT imaging studies have been developed and evaluated for this purpose (Kim et al., 2012; Lee et al., 2013).

Untreated patients with NTM lung disease demonstrate progression of CT findings on serial studies that inform decisions to initiate treatment, particularly with the nodular bronchiectatic form of MAC lung disease (Park et al., 2017). Similar to microbiologic endpoints, radiographic improvement at the conclusion of therapy can characterize successful treatment of NTM lung disease. However, less is known regarding the contribution of early radiographic changes as a marker of treatment response. Moreover, interval radiographic findings during treatment of NTM lung disease may be confounded by the extent of underlying lung disease and by the presence of concomitant infection or colonization of airways (Park et al., 2016; Cohen-Cymberknoh et al., 2017). Other limitations of serial CT imaging for monitoring treatment response include the difficulty with classification of NTM lung disease patients with fibrocavitary disease (an alternate form of MAC lung disease), or structural lung changes due to additional treatment modalities (such as surgical resection or radiation fibrosis) (Lee et al., 2013).

Patients with active chronic lung diseases are exposed to significant radiation when monitored by chest CT imaging. Because proton magnetic resonance imaging (MRI) does not use ionizing radiation this imaging modality has been studied for assessment of cystic fibrosis and sarcoidosis, performing comparably to chest CT in identifying various pathologic patterns of the disease in lung parenchyma. Chung and associates have proposed that MRI may emerge as a viable alternative to CT imaging for assessing the distribution and extent of NTM lung infection (Chung et al., 2016). In an exploratory study of 25 patients known to have lung infection with MAC, they found excellent agreement between noncontrast MRI and CT imaging for detection of nodules and cavities, but only limited agreement for detection of bronchial mucous plugging (Chung et al., 2016; Chung et al. (2016).

fmicb-09-02813 November 17, 2018 Time: 16:32 # 4

**Figure 1**). The authors postulated that further improvement in special discrimination of NTM lung infection may be achievable by application of maximum intensity projection reconstruction and contrast enhancement. To our knowledge, no published studies have reported on the usefulness of MRI for serial assessment of NTM lung infection.

Interval examination with 18F-fluorodeoxyglucose positronemission tomography [18F-FDG PET] is a routine component of monitoring response to chemotherapy for lung cancer, based on the detection of increased glucose metabolism that is characteristic of malignant lesions. More recently, the use of PET modalities has been examined for diagnosis and treatment monitoring of tuberculosis, detecting the increase in glucose metabolism associated with the host inflammatory response (Vorster et al., 2014). Nodular MAC lung disease may show a similar pattern of increased glucose metabolism on PET/CT imaging, raising the possibility of interval monitoring during treatment. Among 6 patients with MAC lung disease assessed with 18F-FDG PET studies before and after MAC-directed treatment, all patients demonstrated a decline in 18F-FDG uptake after treatment, even in the absence of significant improvement in high-resolution CT images (Demura et al., 2009; **Figure 2**).

#### Symptomatic Improvement and Quality-of-Life Monitoring

The concept of clinical cure of NTM disease is tied to symptomatic improvement at the conclusion of treatment, which may not reach complete resolution of symptoms due to underlying structural lung disease (van Ingen et al., 2018).

A number of studies have measured baseline quality-oflife indicators among NTM lung disease patients using various assessment tools, including the St. George Respiratory Questionnaire (SGRQ) (Mehta and Marras, 2011), the EuroQOL Five Dimensions (Hong et al., 2014), and the Medical Outcomes Short-Form-36 Questionnaire (SF-36) (Mehta and Marras, 2011; Asakura et al., 2015). However, there are a paucity of data regarding the formal assessment of quality-of-life during NTM treatment as an interval marker of clinical response, a notable knowledge deficit given the importance of patient reported outcomes in the context of drug development efforts. Importantly, under the 21st Century Cures Act, the FDA has updated the regulatory framework for evaluating these types of patient-reported outcomes in the Clinical Outcome Assessment Qualification Program, which operates in parallel with the Biomarker Qualification Program to support the evaluation of drug development tools for a disease area (FDA, 2018b).

Czaja et al. (2016) studied 47 patients with MAC lung disease, assessing quality-of-life at regular intervals during treatment with the SGRQ, and observing a significant improvement as early as 3 months from the treatment initiation. Notably, improvements in quality-of-life assessments were not related to improvements in CT imaging, perhaps a reflection of the limitations of CT imaging as discussed above, along with the greater degree of overall disease status and complexity that can be captured by quality-of-life assessments. A crosssectional study of MAC complex lung disease patients, based on the SF-36, found that quality-of-life scores were lower among currently treated patients compared to previously- or nevertreated patients, but repeated assessments during treatment were not performed (Asakura et al., 2015). While one interpretation of these findings would be that current MAC treatment modalities negatively affect quality-of-life, an alternate explanation would be that patients are selected for MAC treatment based on current symptoms, as reflected in quality-oflife scores.

#### Blood-Based Monitoring

fmicb-09-02813 November 17, 2018 Time: 16:32 # 5

Both humoral and cell-mediated NTM-driven immune responses have been examined as potential sources of treatment monitoring biomarkers. As an alternative to sputum-based biologic sampling, blood-based monitoring may offer several advantages. Unlike sputum, blood is readily available throughout the treatment course, and continuously distributed biomarkers in blood would support precise comparisons with baseline levels without relying on hierarchical scoring systems (as used for radiographic findings or microbiology results). Furthermore, patients undergoing treatment for extra-pulmonary forms of NTM disease could potentially benefit from a validated blood-based biomarker of treatment response, for example during treatment of deep tissue infections such as osteomyelitis, where clinical response is less apparent on physical examination. Immune-based biomarkers of treatment response may also shed insight into the immunologic correlates of protection against disease in at-risk patient populations.

The Capilia MAC Ab ELISA kit (TAUNS, Shizuoka, Japan) developed for the diagnosis of MAC lung disease, measures serum levels of IgA antibodies against glycopeptidolipid (GPL), a major component of the MAC cell wall (Shibata et al., 2016). Serum anti-GPL IgA levels have been proposed as potential biomarkers of treatment response (Jhun et al., 2017; Kitada et al., 2017), although clinical evidence is limited to studies where serum anti-GPL IgA levels were compared before and after treatment, rather than at earlier timepoints during treatment.

Kitada et al. (2005) followed 27 patients with pulmonary MAC disease, obtaining serum samples before and after anti-MAC therapy. Among patients achieving clinical cure, a reduction in anti-GPL IgA levels was observed, while no difference was observed among 13 patients with treatment failure. Interestingly, a single patient with medical treatment failure then underwent surgical lobectomy, and subsequently demonstrated a fall in serum anti-GPL IgA levels, corresponding to conversion of sputum cultures. In a subsequent study that included 34 patients with MAC lung disease, serum anti-GPL IgA post-treatment declined from pre-treatment values among 19 patients who achieved sputum culture conversion, but not among 7 patients who either had disease recurrence or 8 patients who failed to achieve conversion (Kitada et al., 2017).

Jhun et al. (2017) evaluated serial changes in serum anti-GPL IgA levels during treatment of MAC lung disease at baseline, 3 months, and 6 months of treatment. Across all 57 patients, a decrease in serum anti-GPL IgA levels was observed. In contrast to the earlier findings discussed above (Czaja et al., 2016), patients with unfavorable treatment responses also had significant declines in anti-GPL IgA levels, although baseline levels were higher among patients who eventually developed treatment failure, perhaps reflecting a greater initial disease burden.

Cell-mediated immune responses, rather than antibody responses, could provide an alternate approach for immunologic monitoring of treatment response. Kim et al. (2014) enrolled 42 patients with MAC lung disease and measured serum concentrations of 36 different type I cytokine-associated molecules before and after 12 months of treatment. Declining levels of IL-17 and IL-23 during follow-up were associated with treatment failure, suggesting that continued impairment of the IL-17 pathway reflects ongoing disease processes. These findings are consistent with earlier work demonstrating that low IL-17 levels are a risk factor for developing MAC lung disease (Lim et al., 2010). Depending on the temporal dynamics of serum cytokines in the IL-17 pathway earlier in treatment, the measurement of one or more molecules on this pathway could be of interest as a potential monitoring biomarker.

Carbohydrate antigen 19-9 (CA 19-9) is a treatment monitoring biomarker for several forms of cancer. Elevated blood levels of CA 19-9 are also seen in various forms of chronic lung disease, including bronchiectasis. Recently, CA 19- 9 was examined as biomarker of treatment response among 24 patients with NTM lung disease in South Korea (Hong et al., 2016). Among 17 patients with treatment response (defined as symptomatic improvement and sputum culture conversion within 12 months of therapy), CA 19-9 levels significant declined, while no difference was observed among the 7 patients without clinical response. Larger cohort studies with serial measurements of CA 19-9 earlier in treatment may be of interest.

#### Pulmonary Function Monitoring

Measurement of forced expiratory volume is a prognostic indicator for several chronic respiratory diseases, including COPD and cystic fibrosis, and the FEV1 is a qualified biomarker for COPD drug development efforts. The utility of pulmonary function monitoring as a general biomarker of treatment response for NTM lung disease will likely be limited by the tremendous heterogeneity in underlying chronic lung diseases and NTM disease presentations (for example, nodular bronchiectatic vs. fibrocavitary disease). In a single center study of 37 cystic fibrosis patients with pulmonary M. abscessus infection, early improvements in FEV1 after treatment initiation (either 30 or 60 days) were not associated with longterm microbiologic outcomes or sustained improvements in pulmonary function (DaCosta et al., 2017).

### FUTURE DIRECTIONS

Clinical research centers specializing in the treatment of NTM lung disease will benefit from alignment of prospective data collection schemes that include specimen repositories for blood (both plasma and peripheral blood mononuclear cells), sputum,

and urine. The Bronchiectasis and NTM Research Registry, originally designed to enroll patients with bronchiectasis without underlying cystic fibrosis, supports serial banking of blood and deep respiratory specimens (Aksamit et al., 2017). This framework could provide the basis for investigating some of the potential microbiologic and serum and biomarkers discussed above. Future advances in MRI and PET imaging and in quantitative comparison of serial imaging studies may accelerate future research on changes in anatomic extent and metabolic activity of NTM infections in response to treatment.

Complementary prospective, observational studies would support comprehensive phenotyping of NTM disease patients with serial assessments of novel biomarkers of disease status and treatment response, and potentially could compare treatment monitoring biomarkers between patients treated for pulmonary and extra-pulmonary NTM disease. Prospective studies of novel monitoring biomarkers should also consider the potential for ongoing environmental re-exposure to NTM pathogens leading to re-infection, as can occur with contaminated household water sources (Lande et al., 2018) or nosocomial infections (Baker et al., 2017). Finally, much of the treatment response work thus far has focused on NTM lung disease has centered on MAC, and multicenter enrollment strategies may be essential to study response biomarkers for non-MAC lung disease, in particular lung disease caused by M. abscessus complex. These types of large scale, multicenter studies will also be essential to the prospective evaluation of novel treatment regimens for NTM lung disease. Multiple enrollment sites will be required to provide the statistical power for comparing alternative therapeutic strategies, given the smaller number of NTM lung disease patients compared with tuberculosis on a global scale. Furthermore, clinical studies supported by the National Institutes of Health (Sigal et al., 2017) and the Bill & Melinda Gates Foundation (Chen et al., 2017) have demonstrated the

#### REFERENCES

feasibility of integrating biomarker assessment into clinical trial design.

### SUMMARY

"Quo vadis" ("whither goest thou?") remains a central question for NTM patients and their physicians. It is uncertain whether a single biomarker of clinical response will sufficiently capture the complexity of NTM disease presentations in a heterogeneous patient population, or whether the current "all of the above" strategy can be improved with refinement of microbiologic, radiographic, and quality-of-life assessments. Should a composite biomarker be developed under the FDA Biomarker Qualification program, it would require a "Context of Use" defining the specific algorithm for combining these individual components into a single treatment response measure. Ongoing research efforts for NTM disease should support the evaluation of biomarkers of response to treatment in parallel with studies of novel therapeutics and mechanisms of disease. Patients, clinicians, and investigators will benefit from the knowledge that the road they travel leads toward clinical cure.

### AUTHOR CONTRIBUTIONS

AM, CV, JH-F, and KH performed the literature review. CV wrote the initial draft. AM, HO, RK, JH-F, and KH critically appraised and revised.

### FUNDING

CV was supported by NIH (K23AI10263).

response in patients with pulmonary mycobacteriosis. Eur. J. Nucl. Med. Mol. Imaging 36, 632–639. doi: 10.1007/s00259-008-1009-5

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Vinnard, Mezochow, Oakland, Klingsberg, Hansen-Flaschen and Hamilton. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# A Closer Look at the Genomic Variation of Geographically Diverse Mycobacterium abscessus Clones That Cause Human Infection and Disease

#### Rebecca M. Davidson\*

Center for Genes, Environment and Health, National Jewish Health, Denver, CO, United States

#### Edited by:

Veronique Anne Dartois, Rutgers, The State University of New Jersey, United States

#### Reviewed by:

Joseph Oliver Falkinham, Virginia Tech, United States Rachel Thomson, The University of Queensland, Australia

> \*Correspondence: Rebecca M. Davidson<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

Received: 15 August 2018 Accepted: 19 November 2018 Published: 05 December 2018

#### Citation:

Davidson RM (2018) A Closer Look at the Genomic Variation of Geographically Diverse Mycobacterium abscessus Clones That Cause Human Infection and Disease. Front. Microbiol. 9:2988. doi: 10.3389/fmicb.2018.02988 Mycobacterium abscessus is a multidrug resistant bacterium that causes pulmonary and extrapulmonary disease. The reported prevalence of pulmonary M. abscessus infections appears to be increasing in the United States (US) and around the world. In the last five years, multiple studies have utilized whole genome sequencing to investigate the genetic epidemiology of two clinically relevant subspecies, M. abscessus subsp. abscessus (MAB) and M. abscessus subsp. massiliense (MMAS). Phylogenomic comparisons of clinical isolates revealed that substantial proportions of patients have MAB and MMAS isolates that belong to genetically similar clusters also known as 'dominant clones'. Unlike the genetic lineages of Mycobacterium tuberculosis that tend to be geographically clustered, the MAB and MMAS clones have been found in clinical populations from the US, Europe, Australia and South America. Moreover, the clones have been associated with worse clinical outcomes and show increased pathogenicity in macrophage and mouse models. While some have suggested that they may have spread locally and then globally through 'indirect transmission' within cystic fibrosis (CF) clinics, isolates of these clones have also been associated with sporadic pulmonary infections in non-CF patients and unrelated hospital-acquired soft tissue infections. M. abscessus has long been thought to be acquired from the environment, but the prevalence, exposure risk and environmental reservoirs of the dominant clones are currently not known. This review summarizes the genomic studies of M. abscessus and synthesizes the current knowledge surrounding the geographically diverse dominant clones identified from patient samples. Furthermore, it discusses the limitations of core genome comparisons for studying these genetically similar isolates and explores the breadth of accessory genome variation that has been observed to date. The combination of both core and accessory genome variation among these isolates may be the key to elucidating the origin, spread and evolution of these frequent genotypes.

Keywords: nontuberculous mycobacteria (NTM), genome evolution, pulmonary infection, core genome, accessory genome, Mycobacterium abscessus

### INTRODUCTION

fmicb-09-02988 December 4, 2018 Time: 15:45 # 2

Nontuberculous mycobacteria (NTM) include slowly and rapidly growing species in the genus Mycobacterium that are opportunistic pathogens in humans. A common clinical presentation is pulmonary infection, which occurs in susceptible individuals such as those with cystic fibrosis (CF), COPD, and adults with certain morphological phenotypes (Cassidy et al., 2009; Daley and Griffith, 2010; Winthrop et al., 2010; Martiniano et al., 2017). Another presentation is extrapulmonary NTM infection that can result from environmental exposures or healthcare-associated outbreaks (Lai et al., 1998; Duarte et al., 2009; Li et al., 2017). One of the most clinically challenging NTM that causes human disease is the rapidly growing species, Mycobacterium abscessus (M. abscessus), which is classified into three subspecies: M. abscessus subsp. abscessus (MAB), M. abscessus subsp. massiliense (MMAS) and M. abscessus subsp. bolletii (MBOL). MAB is the most frequently observed subspecies in clinical populations followed by MMAS, and MBOL is relatively rare (Bryant et al., 2016). Epidemiological studies in the United States (US) show an increase in pulmonary NTM infections (including M. abscessus) in CF populations (Olivier et al., 2003; Adjemian et al., 2018) and patients over 60 years old (Prevots et al., 2010). M. abscessus is also one of the most prevalent species associated with NTM infections in Europe (Roux et al., 2009; Hoefsloot et al., 2013). M. abscessus infections are of particular concern due to the bacteria's innate resistance to a range of antibiotics often resulting in lengthy treatment courses and poor clinical outcomes (Jarand et al., 2011; Koh et al., 2011).

An interesting aspect of M. abscessus infections is that little is known about exposure risks and modes of transmission associated with pulmonary infections. The prevailing wisdom is that NTMare acquired from the environment, as they inhabit plumbing and water systems (Feazel et al., 2009; Falkinham, 2011; Ovrutsky et al., 2013; Honda et al., 2016; Zhao et al., 2017), as well as aquatic environments and soil (Primm et al., 2004; De Groote et al., 2006). These surveys have predominantly detected slowly growing NTM species, however, underscoring the potential for M. abscessus to occupy different and possibly yet to be identified niches within the environment. Studies of M. abscessus outbreaks and pseudo-outbreaks have found the offending bacteria in contaminated laboratories (Lai et al., 1998), clinic disinfectants (Tiwari et al., 2003) and hospital water sources (Baker et al., 2017), and some epidemic strains show disinfectant resistance (Duarte et al., 2009; Leao et al., 2010). Environmental surveys of NTM in household environments pinpoint the ecological niche of M. abscessus as indoor water and plumbing biofilms (Thomson et al., 2013a; Honda et al., 2016), M. abscessus also exhibits long-term survival on fomite particles (Malcolm et al., 2017). Epidemiological studies, however, have not found clear links between household exposure risks and acquisition of NTM (Prevots et al., 2014). Only two studies from Australia have demonstrated genetic links between pulmonary patient isolates of M. abscessus and environmental isolates from household water sources (Thomson et al., 2013a,b), though the directionality of infection has not been confirmed. Finally, attempts to isolate M. abscessus from environmental sources in the context of suspected outbreaks within CF centers have been unsuccessful.

The first case of person-to-person transmission of any NTM species was proposed among five CF patients with MMAS cared for at a US CF Center, raising concerns about adequacy of infection control and risks associated with patient exposure during CF care (Aitken et al., 2012). This coincided with using whole genome sequencing (WGS) to characterize genomic diversity and perform genetic strain matching. Since then, several studies have used WGS to examine genetic diversity of pulmonary M. abscessus isolates from patients in a US referral hospital (Davidson et al., 2014), suspected outbreaks of pulmonary infections in CF centers (Bryant et al., 2013; Tettelin et al., 2014; Harris et al., 2015; Tortoli et al., 2017); a nationwide epidemic of soft tissue infections in Brazil (Davidson et al., 2013a; Everall et al., 2017) and a global population study of CF-associated pulmonary isolates from multiple CF centers in three continents (Bryant et al., 2016). One of the most intriguing observations from these studies was that a large proportion of MAB and MMAS clinical isolates grouped into "dominant clones", which have high genetic similarity, but are not identical. Moreover, these clones have been found in nearly every WGS study regardless of worldwide location. This finding led to speculation that the clones may have both spread locally within CF clinics, likely through 'indirect transmission' of contaminated clinical environments, and then globally between CF centers (Bryant et al., 2016). However, detection of these clones sporadically from non-CF patients (Davidson et al., 2014) and in cases of extrapulmonary infections (Ripoll et al., 2009; Everall et al., 2017), combined with their unknown prevalence in the environment, challenge this hypothesis.

This review will take a closer look at the genomic studies of M. abscessus (subspecies MAB and MMAS) and summarize observations of the dominant clones (**Table 1**). It will address the limitations of core genome phylogenomic analysis for genetic matching, and explore how accessory genome variation may be the key to revealing the relationships among clonal isolates from disparate geographic regions and infection types. Genomic studies of M. abscessus will undoubtedly be needed in the future to further study the evolution and spread of the dominant clones in both environmental and clinical settings.

#### GENOTYPING METHODS FOR M. abscessus

Several molecular techniques have been used to identify and classify M. abscessus isolates. Early studies relied on single gene sequencing or multi-locus sequence typing (MLST) using genes such as rpoB (Adekambi et al., 2006), hsp65, secA and the internal transcribed spacer (Adekambi et al., 2003; Adekambi and Drancourt, 2004; Zelazny et al., 2009), to classify isolates to the subspecies level. To further evaluate whether strains were "genetically matched" in the context of potential outbreaks, pulsed field gel electrophoresis (PFGE), randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) and repetitive sequence polymerase chain reaction (rep-PCR)

(Healy et al., 2005) have been used. The highest resolution method for genetic matching is whole genome sequencing (WGS) as millions of genomic positions and thousands of loci can be compared, vs. a limited number of loci (usually less than 100) with the previous methods.

In 2009, a complete reference genome at 5.03 megabases (Mb) was published for the MAB type strain, ATCC19977T (Ripoll et al., 2009) followed by genomes for the type strains of MMAS at 4.8 Mb (CCUG48898T ) (Tettelin et al., 2012) and MBOL at 5.0 Mb (BDT ) (Choi et al., 2012). This ushered in a new era in population genomic studies. Since 2013, several groups have utilized WGS to examine M. abscessus subspecies at the population level, comparing patient isolates within a single clinic (Bryant et al., 2013; Davidson et al., 2014; Harris et al., 2015) and on a larger scale, between CF clinics in one or more countries (Bryant et al., 2016; Tortoli et al., 2017).

#### GENOME STRUCTURE OF M. abscessus

To understand the nuances in interpreting comparative genomics analyses, we should consider the structure of bacterial genomes, which are classified into two components. The **core genome** is defined as the genomic regions or genes that are shared by all isolates within a population (at the genus, species or subspecies level) (Medini et al., 2005). These include "housekeeping genes" that are necessary for survival and which evolve at relatively predictable rates. Variation in the core genome is measured by the single nucleotide polymorphisms (SNP) found in the shared regions. Depending on the species, as much as 65% or more of the genomic space can be analyzed in this way. The **accessory genome** includes genetic material that is shared by one or more, but not all, isolates in a population. This includes regions that are acquired by horizontal gene transfer (HGT) such as genomic islands, transposases, mycobacteriophages, and plasmids, which have the potential to change bacterial phenotypes through acquisition of antimicrobial resistance genes and entire gene cassettes that code for novel metabolic processes (Juhas et al., 2009; Sassi and Drancourt, 2014; Garcia et al., 2015; Gray and Derbyshire, 2018). Accessory genome features, such as genes, groups of genes and plasmids, are measured in an isolate population as presence or absence.

The sum of the core genes plus accessory genes in an isolate population is called the **pan genome**. A pan genome study of 40 M. abscessus genomes from all three subspecies revealed a core genome of 3,345 genes, which is about 68% of a typical MAB or MBOL genome and 71% of a typical MMAS genome (Choo et al., 2014). This means that approximately 30% of the M. abscessus genome is made up of accessory genes that are present in only a subset of isolates or are strain-specific. The accessory genome of M. abscessus is known to include prophages, plasmids and genomic islands transferred by HGT from other bacterial genera (Ripoll et al., 2009; Leao et al., 2013; Davidson et al., 2014; Gray and Derbyshire, 2018) consistent with an environmental bacterium exposed to a diverse microbiome. Population genomic studies can use both core and accessory genome data in their genetic matching analyses, but it is common to find studies using only core genome comparisons as these

have the most well-defined and reproducible computational methods. The accessory genome, however, can provide additional discrimination among closely related isolates.

#### GENOMICS STUDIES OF M. abscessus FROM CF POPULATIONS

Following the first published case of inter-patient transmission of MMAS isolates among CF patients in the US (Aitken et al., 2012), multiple European studies used core genome analysis to look for evidence of person-to-person transmission in their CF clinics (reviewed in detail in Martiniano et al., 2017). A retrospective analysis of 168 M. abscessus isolates from 31 adult CF patients at the Papworth hospital in the United Kingdom (UK) (Bryant et al., 2013) revealed two genetic clusters of MMAS with high genetic similarity. This study was the first to define a quantitative SNP threshold indicating probable transmission between patient isolates as less than 25 SNPs. The use of this SNP threshold for isolates from different patients coupled with social network analysis showed that while the patients lived in different geographic areas, they had multiple opportunities for cross-infection through overlapping clinic visits. Moreover, the potentially transmissible isolates shared the same SNP in the 16S rRNA that confers amikacin resistance, including isolates from patients that had not previously used nebulized aminoglycosides. The authors were unable to identify a local environmental source of MMAS inoculum, and thus surmised that person-to-person transmission did occur within their hospital. The absence of isolation in the environment, however, could also reflect insufficient culturing methods or nonexhaustive sampling. Another study at the Great Ormond Street Hospital in the United Kingdom used a similar experimental design to assess the potential for cross-infection among 20 pediatric CF patients (Harris et al., 2015). Core genome analysis of 27 M. abscessus isolates revealed genetic clusters of both MAB and MMAS. The authors used the suggested threshold of less than 25 SNPs between patient isolates as defined in the Papworth study (Bryant et al., 2013) and identified four patients involved in suspected transmission events, but they did not find intersecting clinic visits between any of the patients. Finally, an Italian study analyzed 162 M. abscessus isolates from 48 patients attending CF centers in "four geographically distinct regions in Italy" (Tortoli et al., 2017). Using a conservative threshold of less than 30 SNPs between patient isolates, they found isolate clusters of MAB, MMAS and MBOL, and identified and seven "possible transmission episodes" among patients. In three potential episodes including two clusters of MMAS and one cluster of MBOL, two patients were found to have attended the same clinic within the same timeframe. The authors concluded that the lack of major outbreaks over the 12-year study period signified minimal risk of inter-human transmission in their CF centers. In summary, the occurrence and frequency of crossinfection between CF patients in the clinic remains controversial and further studies will be needed. It would also be useful to compare and contrast infection control strategies in this context.

In 2016, a large-scale study utilized WGS and core genome analyses to examine the global population structure of 1,080 M. abscessus isolates from 510 CF patients sampled at clinics across multiple European countries, the US and Australia (Bryant et al., 2016). The study identified predominant clusters of genotypes that they coined "dominant circulating clones" as well as several other genetically diverse genotypes. The dominant clones are defined as phylogenetically clustered isolates found in a high proportion of patients. The study identified a primary dominant clone of MAB (known as Abscessus Cluster 1) and a dominant clone of MMAS (known as Massiliense Cluster 1) that corresponds to the "transmissible clone" from the previous Papworth study (Bryant et al., 2013). Collectively, clustered isolates had a higher proportion of drug resistance mutations to amikacin and clarithromycin and were more likely to be associated with chronic infections compared with unclustered isolates. Moreover, the clones had increased phagocytic uptake and intracellular survival in macrophages, and significantly greater bacterial burden and granulomatous inflammation in SCID mice suggesting differences in virulence compared with unclustered isolates.

### DOMINANT CLONE OF M. abscessus subsp. massiliense

Three "transmissible" MMAS isolates from the CF clinic outbreak in Seattle, Washington (Aitken et al., 2012) were sequenced in 2014, and the genomes were compared with other publically available genomes (Tettelin et al., 2014). Intriguingly, the Seattle MMAS isolates were highly similar to the transmissible MMAS clones from the Papworth, United Kingdom CF clinic study (Bryant et al., 2013) and an epidemic MMAS strain from Rio de Janiero, Brazil (CRM-0020) (Davidson et al., 2013a,b). Core genome diversity among the geographically disparate isolates was only 11 to 86 SNPs, and the accessory genome variation included a 11.5 kb genomic island that was unique to the Papworth strains and three genomic regions (totaling 95 kb) and a 44 kb IncP-1β plasmids that were unique to the Brazilian strain. Then, two years later, it was revealed that this potentially "transmissible clone" was the most prevalent MMAS (Massiliense Cluster 1) in the global population study of CF-NTM isolates and was present in all the countries sampled including the United States, United Kingdom, Ireland, Sweden, Netherlands, Denmark, Ireland and Australia (Bryant et al., 2016).

The Brazilian clone (also called BRA100) with high genetic similarity to the pulmonary CF strains was responsible for an epidemic of soft tissue infections from 2004–2009 (Duarte et al., 2009; Leao et al., 2010). The series of infections that spread through multiple states were attributed to contaminated surgical equipment that that had been cleaned with glutaraldehyde, which ultimately selected for a disinfectant resistant clone. A follow up population genomics study of 188 epidemic strains from nine Brazilian states revealed that the isolates had one or two different plasmids (pMAB01 and pMAB02) that were absent from

the corresponding pulmonary CF isolates (Everall et al., 2017). Divergence dating analysis estimated that the Brazilian lineage emerged in 2003 suggesting a recent introduction, however, it is unknown if BRA100 exists locally in the environment of Brazil. The two plasmids from the Brazilian isolates have been fully sequenced (Leao et al., 2013; Everall et al., 2017), but the mechanism of glutaraldehyde resistance is still not known.

#### DOMINANT CLONE OF M. abscessus subsp. abscessus

The dominant clone of MAB identified in the global NTM population study (Abscessus Cluster 1) was found in CF patients from all of the countries and continents sampled (Bryant et al., 2016). Interestingly, this clone is highly genetically similar to the MAB type strain, ATCC19977T , that was initially isolated in the early 1950's from a midwestern US patient with "an acid-fast infection of the knee" and "subcutaneous abscess-like lesions of the gluteal region" (Moore and Frerichs, 1953). This clone was present in pulmonary isolate clusters found in the CF study in Great Ormand Hospital in the United Kingdom (Harris et al., 2015), the M. abscessusstudy from four Italian CF centers (Tortoli et al., 2017), and in a study of pulmonary isolates from CF and non-CF patients in a US referral hospital in Colorado (Davidson et al., 2014).

The MAB dominant clone isolates appear highly similar by core genome SNP analyses, but they also show significant variation in their accessory genomes. For example, the type strain (ATCC19977T ) reference genome includes a 23.3 Kb plasmid with a mercury resistance operon (Ripoll et al., 2009). In the Colorado WGS study with nine clustered pulmonary isolates of the MAB dominant clone, only one (1/9 = 11%) contained the full plasmid, and up to 8.3% of the reference ATCC19977T genome was absent in these isolates including a prophage region and multiple transposase-related genomic islands (Davidson et al., 2014). This study also showed that the nine isolates clustered geographically when comparing the presence or absence of genomic islands suggesting that the

#### REFERENCES

accessory genome may be subject to local, environmental-specific adaptations.

#### CONCLUSION AND PERSPECTIVES

The dominant clones of M. abscessus (including MMAS and MAB) have been observed several times over the years associated with a range of disease etiologies (**Table 1**), Recently, WGS and population genomics has revealed the extent of genomic variation in the core genome with less than 100 variable SNP positions, while the accessory genome can include presence or absence of large plasmids and genomic islands. The widespread geographic diversity of the dominant clones is intriguing, and it is currently not known if they have spread globally via inter-continental transmission or if they inhabitant local environments and just happen to be the most effective genotypes for human infection. A large scale genomic study of randomly sampled environmental NTM isolates would reveal the niches of M. abscessus dominant clones in nature and the true risk of environmental exposure to susceptible patient populations (Strong and Davidson, 2017). Such environmental studies are time consuming and costly, and appropriate funding mechanisms will need to be identified. Future genomic studies of M. abscessus will benefit from analyzing diversity in both the core and accessory genes to reveal local adaptations, identify potential mechanisms of virulence and monitor the evolution and mutation rates of these clinically important genotypes.

#### AUTHOR CONTRIBUTIONS

RD conceived of topic and wrote the manuscript.

#### FUNDING

RD acknowledges the NIH/NIAID (award no. 5K01AI125 726-02).

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Davidson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Global Environmental Nontuberculous Mycobacteria and Their Contemporaneous Man-Made and Natural Niches

Jennifer R. Honda1 \*, Ravleen Virdi1 and Edward D. Chan2,3,4

1 Department of Biomedical Research and the Center for Genes, Environment, and Health, National Jewish Health, Denver, CO, United States, 2 Medicine and Academic Affairs, National Jewish Health, Denver, CO, United States, 3 Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Denver, Aurora, CO, United States, 4 Department of Medicine, Denver Veterans Affairs Medical Center, Denver, CO, United States

#### Edited by:

Thomas Dick, Rutgers, The State University of New Jersey, Newark, United States

#### Reviewed by:

Joseph Oliver Falkinham, Virginia Tech, United States Giovanni Delogu, Università Cattolica del Sacro Cuore, Italy

> \*Correspondence: Jennifer R. Honda<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

Received: 26 June 2018 Accepted: 10 August 2018 Published: 30 August 2018

#### Citation:

Honda JR, Virdi R and Chan ED (2018) Global Environmental Nontuberculous Mycobacteria and Their Contemporaneous Man-Made and Natural Niches. Front. Microbiol. 9:2029. doi: 10.3389/fmicb.2018.02029 Seminal microbiological work of environmental nontuberculous mycobacteria (NTM) includes the discovery that NTM inhabit water distribution systems and soil, and that the species of NTM found are geographically diverse. It is likely that patients acquire their infections from repeated exposures to their environments, based on the well-accepted paradigm that water and soil bioaerosols – enriched for NTM – can be inhaled into the lungs. Support comes from reports demonstrating NTM isolated from the lungs of patients are genetically identical to NTM found in their environment. Well documented sources of NTM include peat-rich soils, natural waters, drinking water, hot water heaters, refrigerator taps, catheters, and environmental amoeba. However, NTM have also been recovered in biofilms from ice machines, heated nebulizers, and heater-cooler units, as well as seat dust from theaters, vacuum cleaners, and cobwebs. New studies on the horizon aim to significantly expand the current knowledge of environmental NTM niches in order to improve our current understanding of the specific ecological factors driving the emergence of NTM lung disease. Specifically, the Hawaiian Island environment is currently being studied as a model to identify other point sources of exposure as it is the U.S. state with the highest number of NTM lung disease cases. Because of its geographic isolation and unique ecosystem, the Hawaiian environment is being probed for correlative factors that may promote environmental NTM colonization.

Keywords: nontuberculous mycobacteria, environments, man-made, natural, Hawaii

### BACKGROUND

Nontuberculous mycobacteria (NTM) and their environments are intricately bound. NTM share these environments with humans and domesticated animals and repeated exposure is a wellaccepted mode of acquiring these infections. While most casual NTM exposures do not results in disease, those with anatomic lung abnormalities of bronchiectasis and emphysema are particularly predisposed to develop NTM lung disease (NTM-LD). However, in individuals without obvious pre-existing risk factors, it is likely that multiple risk factors – some genetic or other acquired factors – may collude to increase their vulnerability. While there have been case reports and small case series linking genetically identical NTM in patients to their home environment, the

specific factors facilitating their acquisition remains poorly characterized. These factors encompass (i) varied sources of infection, (ii) modes of aquisition, and (iii) other physical aspects of the environment such as temperature, humidity, air exchange, surface types, and turbulence created by wind and natural disasters, and (iv) human behaviors, the combination of which are likely to be relatively unique among affected individuals (Honda et al., 2015; Nishiuchi et al., 2017).

Herein, we discuss the traditional environmental niches associated with NTM organisms, but also review the lesser-recognized environmental locales that NTM colonize including non-traditional niches and non-human hosts. We also summarize previous studies that link ecological factors with risk for infection and epidemiological information. Finally, we introduce new, ongoing work to study the particular environmental drivers of NTM emergence in Hawai'i, a geographic location deemed a major hot spot for NTM-LD.

### NTM ORIGINS?

The interaction of humans with their natural and built environments along with changes humans make to their environment (e.g., installation of mechanical devices that change environmental temperature and humidity, or others), and differences in the robustness of human health may impact the emergence of infectious diseases. The origins of NTM-LD remain a mystery, but may be intrinsically linked to human interaction with their environments. As soon as early humans learned to prepare small, controlled fires, the light and heat produced gathered people together for social interactions, vastly improved food preparation, and helped communities defend against invasion – activities that significantly lengthened human survival (Chisholm et al., 2016). Because NTM are found in the environment, it is plausible that these bacteria were historically aerosolized from soil with increased fire-making, infecting human lungs already impacted by long-term exposure to campfire smoke. Nonetheless, there is no evidence to show that the number of NTM-LD cases increase after camping or large scale environmental fires. For now, the origins of lung disease caused by NTM organisms remains an area of investigation.

### GLOBAL GEOGRAPHY AND NTM

The ability of NTM to cause LD and its clinical relevance varies globally. Geographic distribution of NTM species provides information regarding geographic-specific drivers of exposure such as climate, environment, and host factors associated with NTM-LD that would be specific to a global region (Falkinham, 1996; Griffith et al., 2007).

Mycobacterium avium complex (MAC) is the most frequently isolated group of NTM species worldwide and the most common organism associated with NTM-LD. MAC consists of various species of slow-growing mycobacteria (SGM) including M. avium, M. intracellulare, M. chimaera, M. colombiense, M. marseillense, M. arosiense, M. timonense, M. bouchedurhonense, and M. ituriense. Subspecies of M. avium include avium, silvaticum, hominissuis, and paratuberculosis (Hoefsloot et al., 2013; Johnson and Odell, 2014). Currently, the lowest number of MAC isolates in the world are seen in South America, but this may be because there is little NTM information available from this region (Hoefsloot et al., 2013; Halstrom et al., 2015; Stout et al., 2016). High incidence of NTM-LD due to MAC, M. kansasii, M. gordonae, and M. malmoense are observed in Europe, North America and Australia.

Rapid-growing mycobacteria (RGM) including the Mycobacterium abscessus and Mycobacterium fortuitum groups also contribute to a large proportion of NTM-LD cases globally. The Mycobacterium abscessus group is comprised of three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii. In the United States (U.S.), M. abscessus complex infections are secondary only to MAC infections, comprising 3-13% of all NTM-LD cases (Lee et al., 2015). The M. abscessus group is also commonly observed in patients in East Asia; in Taiwan 17.2% of all clinical NTM isolates belong to the M. abscessus group (Lai et al., 2010). The M. fortuitum group comprises M. fortuitum, M. peregrinum, M. senegalense, M. alvei, M. houstonense, M. neworleansense, M. boenickei, M. septicum, and M. porcinum (Adekambi and Drancourt, 2004; Schinsky et al., 2004). Besides LD, M. fortuitum cause soft tissue, skeletal, catheter-related, and disseminated infections in immunocompromised patients (Brown-Elliott and Wallace, 2002).

### United States

The most commonly occurring NTM species in many parts of U.S. are MAC and M. kansasii (Hoefsloot et al., 2013). Nearly 80% of all NTM-LD in the U.S. is due to a species of MAC, followed by M, kansasii - the second most common NTM associated with LD (Falkinham, 1996; Griffith et al., 2007). The U.S. states with the highest overall risk for NTM-LD include Hawai'i, California, New York, Louisiana, Pennsylvania, Florida, Oklahoma and Wisconsin (Adjemian et al., 2012a). M. abscessus is the most commonly recovered RGM from southeastern parts of U.S. (Halstrom et al., 2015; Stout et al., 2016). For unknown reasons, NTM-LD cases are lowest in North Dakota, South Dakota, Minnesota, Michigan, New Mexico, and West Virgina (Adjemian et al., 2012a).

### Europe and United Kingdom