key: cord-0701725-bqm05lsr
authors: Dohál, Matúš; Porvazník, Igor; Solovič, Ivan; Mokrý, Juraj
title: Whole Genome Sequencing in the Management of Non-Tuberculous Mycobacterial Infections
date: 2021-10-27
journal: Microorganisms
DOI: 10.3390/microorganisms9112237
sha: 6a6864fb03dbf284c3ce5a22da3e7ca1e77a53f8
doc_id: 701725
cord_uid: bqm05lsr

Infections caused by non-tuberculous mycobacteria (NTM) have been a public health problem in recent decades and contribute significantly to the clinical and economic burden globally. The diagnosis of infections is difficult and time-consuming and, in addition, the conventional diagnostics tests do not have sufficient discrimination power in species identification due to cross-reactions and not fully specific probes. However, technological advances have been made and the whole genome sequencing (WGS) method has been shown to be an essential part of routine diagnostics in clinical mycobacteriology laboratories. The use of this technology has contributed to the characterization of new species of mycobacteria, as well as the identification of gene mutations encoding resistance and virulence factors. Sequencing data also allowed to track global outbreaks of nosocomial NTM infections caused by M. abscessus complex and M. chimaera. To highlight the utility of WGS, we summarize recent scientific studies on WGS as a tool suitable for the management of NTM-induced infections in clinical practice.

Non-tuberculous mycobacteria (NTM) are all species of bacteria within the genus Mycobacterium (M.), except M. tuberculosis and M. leprae; so far, more than 180 species have been identified [1] . NTM are environmental, opportunistic pathogenic bacteria and only a limited number of species cause infections that primarily affect the lungs (pulmonary disease), skin, soft tissues, lymph nodes, but are also responsible for surgical wound infections, implant-associated and catheter infections [2] [3] [4] . The pulmonary form of the disease may be manifested by hypersensitivity pneumonitis, nodular bronchiectasis, or fibrovascular disease. Infections caused by NTM have risen rapidly in recent decades, and in many developed countries, the number of cases has exceeded the incidence of pulmonary tuberculosis [5] . In addition, some species of mycobacteria are also associated with increased mortality in domestic, wild animals, or aquatic organisms, thus contributing to high economic damage and negative environmental impact [6] [7] [8] .

Species belonging to the M. avium complex (M. chimaera, M. intracellulare, M. avium) are the most widespread globally, but species within M. abscessus complex (M. abscessus subsp. abscessus, M. abscessus subsp. boletti, M. abscessus subsp. massiliense) also pose a great threat due to high degree of resistance [9, 10] . The increasing incidence of infections caused by NTM is associated with demographic changes, aging population, and an increasing number of patients with chronic diseases (e.g., chronic obstructive pulmonary disease, cystic fibrosis (CF), cancer, diabetes), including immunosuppressive conditions [11] . However, the disease can also occur in patients without obvious predisposition, which may be related to specific gene mutations encoding changes in the function of the immune system [12] . laboratories in countries with a high incidence of tuberculosis, the diagnosis of mycobacterial infections is based mainly on the culture detection of acid-fast bacilli in a sputum sample and chest radiograph (X-ray or high-resolution computed tomography findings), which complicates the differentiation of NTM from M. tuberculosis complex due to morphological similarities together with identical clinical symptoms, and can lead to incorrect and ineffective treatment regimen [26] . In this context, Advani et al. performed a WGS study on M. tuberculosis isolates and found that almost 20% of all isolates contained NTM mycobacteria, indicating coinfection [27] . Ongoing NTM infection symptoms are often non-specific and can be also misdiagnosed as chronic obstructive pulmonary disease, chest infection of unknown cause, or, more recently, COVID-19. Therefore, the correct species-level identification of pathogens is essential in clinical practice.

WGS technologies provide the possibility of taxonomic reconstruction and identification of unique families of genes occurring within the genus Mycobacteria with biomedical application in the development of new reliable diagnostic tools [1] . These data are also useful in identifying rare NTM, the clinical significance of which is greatly underestimated, because they remain unrecognized in diagnostic laboratories, highlighting the utility of WGS compared to traditional diagnostic methods [28] . In general, NTM colonies grown on solid media (e.g., Lowenstein-Jensen) or in liquid media (Middlebrook 7H9 broth) of automated incubation system (BACTEC MGIT 960 system, Becton, Dickinson and Company, Franklin Lakes, NJ, USA; VersaTREK system, Thermo Fisher Scientific Waltham, MA, USA; MB/BacT Alert 3D, bioMérieux, Marcy-l'Étoile, France) proceeded from clinical (pulmonary-sputum; extrapulmonary-blood, tissues) or environmental samples (microbiological swabs) are the most commonly used materials for diagnostic and sequencing purposes ( Figure 1 ). 

The early symptoms of NTM disease are usually non-specific. Patients complain of chest pain, productive cough, shortness of breath, fatigue, and fever [25] . In clinical laboratories in countries with a high incidence of tuberculosis, the diagnosis of mycobacterial infections is based mainly on the culture detection of acid-fast bacilli in a sputum sample and chest radiograph (X-ray or high-resolution computed tomography findings), which complicates the differentiation of NTM from M. tuberculosis complex due to morphological similarities together with identical clinical symptoms, and can lead to incorrect and ineffective treatment regimen [26] . In this context, Advani et al. performed a WGS study on M. tuberculosis isolates and found that almost 20% of all isolates contained NTM mycobacteria, indicating coinfection [27] . Ongoing NTM infection symptoms are often nonspecific and can be also misdiagnosed as chronic obstructive pulmonary disease, chest infection of unknown cause, or, more recently, COVID-19. Therefore, the correct specieslevel identification of pathogens is essential in clinical practice.

WGS technologies provide the possibility of taxonomic reconstruction and identification of unique families of genes occurring within the genus Mycobacteria with biomedical application in the development of new reliable diagnostic tools [1] . These data are also useful in identifying rare NTM, the clinical significance of which is greatly underestimated, because they remain unrecognized in diagnostic laboratories, highlighting the utility of WGS compared to traditional diagnostic methods [28] . In general, NTM colonies grown on solid media (e.g., Lowenstein-Jensen) or in liquid media (Middlebrook 7H9 broth) of automated incubation system (BACTEC MGIT 960 system, Becton, Dickinson and Company, Franklin Lakes, NJ, USA; VersaTREK system, Thermo Fisher Scientific Waltham, MA, USA; MB/BacT Alert 3D, bioMérieux, Marcy-l'Étoile, France) proceeded from clinical (pulmonary-sputum; extrapulmonaryblood, tissues) or environmental samples (microbiological swabs) are the most commonly used materials for diagnostic and sequencing purposes ( Figure 1 ). Figure 1 . Schematic representation of the WGS methodological procedure. In the first step, DNA is extracted from colonies grown on a solid or liquid culture medium. Subsequently, sequencing libraries are prepared for short-or long-read sequencing. In the last step, the sequencing data are bioinformatically processed for genotyping, identification of resistance patterns, molecular-epidemiology analysis, and assembling of complete genomes of new NTM species. In the first step, DNA is extracted from colonies grown on a solid or liquid culture medium. Subsequently, sequencing libraries are prepared for short-or long-read sequencing. In the last step, the sequencing data are bioinformatically processed for genotyping, identification of resistance patterns, molecularepidemiology analysis, and assembling of complete genomes of new NTM species.

Quan et al. tested the efficacy of WGS for NTM classification compared to conventional available line probe assays (GenoType CM test, GenoType Mycobacterium MTBC or GenoType Mycobacterium AS test). Their results confirmed the high agreement of the individual methods, of which sequencing showed the highest discriminatory power, including within complex diversity (important especially within M. avium complex and M. abscessus complex) and identification of rare species, for example, M. ratisbonense or M. tomidae [29] . This is due to the limited number of amplified genes included in the conventional assays in combination with higher homology between mycobacterial species in contrast with other bacterial species [30] . The disadvantage of WGS compared to line probe assay is a slightly longer turnaround time (3-15 days) . Similarly, the study by Yoon et al. confirmed 100% compliance of WGS and classical molecular genetic methods (PCR and direct sequencing) in the diagnostics of M. avium subsp. hominissuis, M. intracellulare, and M. abscessus [21] .

In addition, WGS has proven to be a suitable tool to distinguish between relapse and reinfection of NTM caused predominantly by M. ulcerans or M. abscessus [31, 32] . WGS also allows the identification of NTM at the clone level, thus allowing the form (relapse/reinfection) of the ongoing disease to be determined [33] . Flohr et al. have applied WGS in diagnostics of factitious disorders caused by NTM that may be misdiagnosed as a recurrent or chronic forms of infection [34] . The need to correctly clarify the form of the disease is highlighted by the high recurrence rate of some NTM infections, which is up to 10-48% after successful treatment [35, 36] .

Accurate diagnosis of NTM is crucial in patients with structural or inflammatory lung disease such as CF, non-cystic fibrosis bronchiectasis, or chronic obstructive pulmonary disease, as these emerging pathogens cause an accelerated decline in lung function and do not respond to aggressive antibiotic treatment in up to 50% of cases [37] . The most important predisposing factors in CF patients include nutritional condition, presence of bronchiectasis, and impaired mucociliary clearance [38] . In addition, the prevalence of NTM in CF patients is almost 10,000-fold higher compared to the rest of the population [39] . The two most common NTM species isolated in CF belonging to M. avium complex (representing 75% of NTM infections in CF) and M. abscessus complex (most common M. abscessus subsp. abscessus and M. abscessus subsp. massiliense) with prevalence ranges from 3 to 23% in CF centers worldwide [40] . The WGS of M. abscessus ATCC19977 showed that this species encoded virulence factors typical of both mycobacterial pathogens and common CF pathogens (Pseudomonas aeruginosa and Burkholderia cepacia) [41] . Trovato et al. confirmed that WGS can more effectively discriminate M. abscessus subspecies in patients with CF in comparison with a variable number of tandem repeat (VNTR) methods [42] . Similar results were shown in a recent study by Redondo et al. in which they successfully diagnosed individual subspecies of M. abscessus in CF-and non-CF patients [43] . Furthermore, M. abscessus complex infections are seen as a contraindication to lung transplant in some CF patients (currently the only cure for CF), due to the high likelihood of re-infection [44] . The risk of disseminated post-transplant infection is potentiated in patients with a positive presence of M. abscessus in sputum before lung transplantation [45, 46] . Kavaliunaite et al. confirmed the association between a particular M. abscessus sequence type (ST) and the success of heart and lung transplantation in CF patients [47] . These results are particularly important as reported cases are increasing, and in some studies, the rate of NTM infections in lung transplants was up to 8% and was directly related to an increased risk of death [48, 49] . In contrast, post-transplant infections caused by M. avium complex are less frequent and associated with a higher cure rate [46] . To decrease the risk of mortality, clinicians should consider the pretransplant or posttransplant (colonized by rapidly-growing NTM) chemoprophylaxis in all patients undergoing the lung transplant [50] .

The benefit of WGS has also been shown in the management of multiple-NTM infections, the diagnosis of which is necessary for the correct setting of the treatment regimen and outcome expectations, as well as a better understanding of species diversity in various anatomical regions of the lung [51] . A recent WGS study by Shaw et al. analyzing the genomes of M. abscessus isolates revealed the multiple subpopulations diversity with different antimicrobial resistance profiles in children with CF. The results of this study are also important for the clinical diagnosis of the disease, as they confirmed a higher diversity of M. abscessus in samples of lungs, chest wounds, and pleural fluid compared to sputum [52] . Sequencing data provided a more detailed subspecies classification in mixed cultures compared to conventionally used methods [53, 54] . WGS also contributed to the finding of a high interspecies and intraspecies M. avium complex diversity in a patient with an ongoing pulmonary form of the disease. These results elevate the clinical potential of WGS, as commercial probes resolve M. avium species only to the complex level [55] . Greninger et al. also used WGS technology to diagnose polymicrobial infection in brain abscess caused by rare, rapidly growing M. immunogenum and M. llatzerense [56] .

For diagnostic purposes, better treatment results, and expanding our knowledge of mycobacterial diseases, it is necessary to identify and classify new species/subspecies of NTM [28, 57, 58] . Describing the complete genomes of the novel NTM requires a combination of short-read (e.g., Illumina MiSeq, San Diego, CA, USA) and long-read (e.g., MinION, Oxford, UK; PacBio SMRT, Pacific Biosciences, CA, USA) sequencing technologies ( Figure 1 and Table 1 ) [59] . Short-read sequencing is characterized by high accuracy but does not provide information about the complete genome (including G + C rich regions, recombination, repetitive PE/PPE regions, deletions, and insertions) [60] . In contrast, long-read sequencing technologies have increased read lengths 100-to 1000-fold with slightly lower accuracy compared to NGS platforms and can span much larger repeat regions than NGS, thus contributing to new genome assembly [61] . Matsumoto et al. identified 27 genomes belonging to the novel NTM. In addition, sequencing data obtained using long-read sequencing technology were used in the same study to develop a multi-locus sequence typing database and software to identify mycobacteria. Sensitivity and specificity were highest compared to conventional methods in the characterization of 29 species of NTM [62] . Hendrix et al. combined Illumina and MinION sequencing for whole-genome assembling of M. kubicae isolates. Their results have provided detailed information regarding the resistance, virulence and persistence encoded on the chromosome or plasmids of these rare, clinically important pulmonary disease-causing NTM [63] . A recent study showed that MinION sequencing technology can be used to detect mycobacteria directly from clinical samples and diagnose the disease within 20 min, therefore setting the treatment regimen in a clinically relevant time frame [64] .

Although NTM resistance is becoming a growing threat, susceptibility testing in clinical laboratories is performed primarily by broth microdilution and macrodilution phenotypic methods which may be difficult to evaluate [65] . Additionally, the treatment for NTM infections usually includes a combination of macrolide antibiotics (such as azithromycin and clarithromycin) with aminoglycosides (for fast-growing NTM) or first-line antituberculosis drugs (for slow-growing NTM). However, for some combinations, clinical breakpoints are not well defined, therefore, the effectiveness of treatment can be greatly reduced [5, 66] . Generally, many NTM species are naturally resistant to specific drugs, however, improperly adjusted and prolonged treatment regimen leads to the development of acquired resistance in originally drug-sensitive bacterial strains [67] . A key factor that plays a role in natural NTM resistance is the low permeability of the mycobacterial cell wall [68] . However, M. abscessus exhibits intrinsic high-level resistance to ethambutol and fluoroquinolones. This resistance is predominantly associated with mutations in the embB (encoded resistance to ethambutol) and gyrA (encoded resistance to fluoroquinolones) genes [69] . M. abscessus and M. avium exhibit intrinsic resistance to rifampicin, primarily due to mutations in the rpoB gene and the presence of the MAB_0591 gene [65] . Another important mechanism of intrinsic resistance is the overexpression of efflux pumps, which is responsible for the resistance of M. abscessus, M. avium, and M. intracellulare to bedachilin and clafazimine [70] [71] [72] . Acquired drug resistance in mycobacteria is often mediated by a chromosomal mutation in genes encoding targets [73] . With the utility of WGS, it is possible to describe the underlying mechanisms behind the drug resistance of NTM species and increase the effectiveness of treatment regimens [74] . Wetzstein et al. studied the efficacy of WGS in predicting M. abscessus resistance to macrolides and aminoglycosides. The results obtained by WGS were shown to be in full agreement with the Genotype NTM-DR line probe assay and phenotypic drug susceptibility testing [75] . Lipworth et al. used sequencing data obtained from M. abscessus species for the identification of new mutations in erm and rrs genes potentially associated with macrolide antibiotic resistance. These mutations are not currently included in traditional genotyping tests (such as GenoType NTM-DR; Hain Lifescience, Nehren, Germany) which may therefore show false-negative results [76] . Similarly, Chen et al. performed WGS on clofazimine-resistant strains of M. abscessus. Their results revealed several high-confidence gene mutations (in MAB_2299c, MAB_1483, and MAB_0540 genes) involved in resistance to this drug, whose use is increasingly preferred due to limited treatment options [77] . In addition, M. abscessus subsp. abscessus possesses the erm gene, while M. abscessus subsp. massiliense harbor the nonfunctional variant of this gene that lacks the inducible resistance phenotype, so it is important in clinical practice to distinguish between these two subspecies [62] . In contrast, a recent WGS study involving a global collection of M. abscessus complex isolates revealed a 10% higher frequency of mutations in the rrl gene encoding macrolide resistance in M. massiliense compared to M. abscessus subsp. abscessus [78] . Based on WGS-data, Yoshida et al. developed special DNA-chromatographic and PCR-based diagnostic assays for discriminating macrolideresistant/susceptible subspecies of M. abscessus (M. massiliense, M. abscessus, M. bolletii). The agreement rate with WGS was 99.7% [79] . Realegano et al. developed a novel clinical whole genome sequencing assay to determine the resistance of M. abscessus to clarithromycin and amikacin. The accuracy of resistance prediction compared to phenotypic results was 100% for both drugs [80] .

However, the increased incidence of resistant strains is leading to the development of new treatment strategies such as treatment with lytic phages. Dedrick et al. used WGS for a better understanding of phage resistance mechanisms of M. abscessus [81] .

Nosocomial NTM infections have been defined as infections directly associated with a hospital stay, or their symptoms may appear immediately after discharge [82] . The first cases of nosocomial NTM infections caused by M. fortuitum have been recorded since the beginning of the 20th century [83] . Nowadays, NTMs are classified as welladopted nosocomial pathogens with a wide range of clinical manifestations and hospital sources of infection [26] . Their adaptation to the hospital environment results from their resistance to disinfectants and sterilants, low nutrient requirements, and ability to form a biofilm [2] . These nosocomial infections are usually extrapulmonary: bloodstream infections, postoperative wound infections, implant-associated infections, spinal infections, etc. [84] .

Genotyping of individual NTM strains is crucial for tracing the nosocomial outbreaks and epidemiological investigation. Previously preferred typing methods, including biotyping, serotyping, and antibiograms, are no longer performed in reference laboratories due to their insufficient discriminatory power. Current genotyping techniques based on 16S rRNA sequencing, pulsed-field gel electrophoresis, VNTR, and spoligotyping or plasmid typing, are relatively easy to perform in a short time frame and require only equipment available in most laboratories [85] . However, these techniques show some limitations in recognizing similarities and differences between close phylogenetically similar strains, which are reflected in the misalignment of these strains within clusters and by the inability to identify their common ancestor [86, 87] . Recent studies confirmed that WGS exhibits higher discriminatory power and a wide range of uses in molecular-epidemiology analysis than current genotyping techniques ( Figure 2) [88, 89] . The results of Harris et al. confirmed higher resolution of WGS in comparison with VNTR profiling in distinguishing NTM subspecies into individual clades [90] . The WGS phylogenomic analysis is usually based on the identification of a massive number of different single nucleotide variants between isolates to cluster and compare genomes, infer relatedness and identify the source of infection. A factor complicating the diagnosis of the disease as well as the tracing for the source of the infection is the long incubation period, which can last several months or years [91] . erative wound infections, implant-associated infections, spinal infections, etc. [84] .

Genotyping of individual NTM strains is crucial for tracing the nosocomial outbreaks and epidemiological investigation. Previously preferred typing methods, including biotyping, serotyping, and antibiograms, are no longer performed in reference laboratories due to their insufficient discriminatory power. Current genotyping techniques based on 16S rRNA sequencing, pulsed-field gel electrophoresis, VNTR, and spoligotyping or plasmid typing, are relatively easy to perform in a short time frame and require only equipment available in most laboratories [85] . However, these techniques show some limitations in recognizing similarities and differences between close phylogenetically similar strains, which are reflected in the misalignment of these strains within clusters and by the inability to identify their common ancestor [86, 87] . Recent studies confirmed that WGS exhibits higher discriminatory power and a wide range of uses in molecular-epidemiology analysis than current genotyping techniques ( Figure 2) [88, 89] . The results of Harris et al. confirmed higher resolution of WGS in comparison with VNTR profiling in distinguishing NTM subspecies into individual clades [90] . The WGS phylogenomic analysis is usually based on the identification of a massive number of different single nucleotide variants between isolates to cluster and compare genomes, infer relatedness and identify the source of infection. A factor complicating the diagnosis of the disease as well as the tracing for the source of the infection is the long incubation period, which can last several months or years [91] . In the past, it was believed that NTM were transmitted to humans only after exposure to the environment, including soil particles or water droplets containing mycobacteria. The ability of NTM to survive and proliferate in this environment is provided by a lipidrich and triple-layered outer membrane that protects the cells against acids, high temperatures, and ultraviolet light [2] . However, WGS served to shift this paradigm by allowing the characterization of patient-to-patient transmission events and contributing to an increase in reports of outbreaks of nosocomial NTM infection. In 2012, Aitken et al. reported In the past, it was believed that NTM were transmitted to humans only after exposure to the environment, including soil particles or water droplets containing mycobacteria. The ability of NTM to survive and proliferate in this environment is provided by a lipid-rich and triple-layered outer membrane that protects the cells against acids, high temperatures, and ultraviolet light [2] . However, WGS served to shift this paradigm by allowing the characterization of patient-to-patient transmission events and contributing to an increase in reports of outbreaks of nosocomial NTM infection. In 2012, Aitken et al. reported person-to-person transmission of M. abscessus subsp. massiliense among the patients with CF for the first time [92] . Further studies followed documenting healthcare-associated cross-transmission of M. abscessus between CF patients, including pediatric patients in national CF centers (with less than 7 SNP difference between isolates) [25, [93] [94] [95] [96] . In many of these studies, the specific lineage was isolated only from patients and not from environmental sources. Moreover, Bryant et al. performed a WGS-based study of a collection of M. abscessus isolates obtained from worldwide (Europe, Australia, the Republic of Ireland, and the United States) community of CF patients (1080 isolates from 510 CF patients). The results confirmed the global circulation of three dominant, genetically similar, multi-drug resistant M. abscessus clones (74% isolates were clustered) that are transmitted within CF clinics [97, 98] . These clones are associated with poorer clinical outcomes and showed increased intracellular survival, pathogenicity, and virulence in macrophage and mouse models [99] . Interestingly, the study of Lipworth et al. showed that most genetically related M. abscessus clones isolated from CF patients had no identifiable relevant epidemiological traceback [100] . In addition, Bronson et al. performed a comparative genomic analysis of 1279 M. abscessus complex genomes from CF and non-CF patients. The results of the study identified small pairwise SNP distances and similar phylogenetic patterns between isolates from patients with CF and without CF, suggesting that these cases originate from a recent common ancestor [77] . However, how these clones are widespread worldwide, either by asymptomatic transmission or by the environment, has not yet been confirmed. Nevertheless, the prevention and control guidelines recommend isolating infected patients to prevent further transmission of M. abscessus within healthcare facilities [101] .

In addition to M. abscessus, the transmission of M. avium complex (predominantly M. chimaera) was also studied. A recent study defining the genetic relationship of M. avium complex isolates between patients and between patients and their environment confirmed the minimal likelihood of patient-to-patient transmission and identified the hospital water distribution system as the main reservoir of NTM [21] . Moreover, WGS has contributed to the characterization of the global outbreak of nosocomial infections caused by M. chimaera in patients who underwent cardiac surgery. These infections are usually presented as prosthetic valve endocarditis, disseminated infections, or infections of vascular grafts [102] . The first outbreak investigation began in 2013 at the University Hospital Zurich in patients with confirmed presence of M. chimaera after cardiac surgery [103] . WGS epidemiological analysis in other studies demonstrated that aerosols from the water tanks in the heatercooler units contaminated with M. chimaera can be airborne disseminated to patients and the surrounding environment [87, [104] [105] [106] [107] [108] . In this context, Götting et al. found the genetically related M. chimaera contaminated the air of the heater-cooler units as well as medical instruments in the operating room [109] . Most of the reported nosocomial outbreaks included patients who were exposed to heater-cooler units from the same manufacturer during the open-heart surgery (LivaNova, London, United Kingdom). A subsequent study confirmed the culture positivity of M. chimaera in water samples on the manufacturer's site and thus confirmed the source of infection [107] . Despite the aggressive therapy with the combination of at least 3 antibiotics (usually including the ethambutol, rifamycin, and macrolide) and surgical removal of any involved devices, 50% of the patients died from complications associated with M. chimaera infection. This is due to the ability of M. chimaera to replicate in the tissues and disseminate to other organs even with previous long antibiotic therapy [110] . The Centers for Disease Control and Prevention (CDC, Atlanta, GA, USA) has also recently released guidelines for the management and diagnostics of heater-cooler unit-associated NTM infections, in which WGS plays a crucial role [111] . The available data also suggest that the risk of M. chimaera infection in patients after cardiac surgery is almost identical to the level of risk of infection in HIV-positive patients [112] . Moreover, clinical symptoms and laboratory findings are often nonspecific, therefore, every patient with a diagnosis of sarcoidosis or culture-negative endocarditis after exposure to a heatercooler unit should be considered as M. chimaera positive [113] . Surgical site infection by M. chimaera may also occur, predominantly in patients after plastic surgery. WGS showed the genetic relatedness of isolates from patients and the samples of tap water at the surgical clinic, therefore exposure of the wound to any non-sterile water should be totally forbidden before complete wound healing [114] . Insufficient sterilization of surgical instruments and water has been suggested as a source of M. chimaera in many other studies, highlighting the utility of WGS in clinical practice [115, 116] .

Labuda et al. used the WGS approach for characterization of a new species of rapidly growing NTM (Mycobacterium FVL 201832) and identification of saline flushes as a source of nosocomial bloodstream infections in oncology patients. These results contributed to the consideration of new state regulations to monitor and reduce the spread of NTM in oncology clinics [117] . Adherence to these regulations is essential, as recent studies have confirmed that M. avium complex infection increases tumor-genes inflammatory responses which could lead to the development of lung and breast cancer and other complications in oncological patients [118] . Similarly, a study by Inkster et al. revealed hospital water supply contaminated with M. chelonae as a potential source of infection in hemato-oncology patients [119] . The genetic diversity of unique species of M. shigaense (belonging to M. simiae complex) causing cutaneous infections have been studied using WGS technology [120] .

NTM infections are associated with a substantially impaired quality of life, increased morbidity, and mortality, and high treatment costs, therefore, it is essential to use sufficiently sensitive diagnostic methods such as WGS. Even though the cost of WGS samples decreases every year, the implementation of this method in clinical diagnostic laboratories requires a higher initial capital and well-trained personal therefore is not available in the mycobacteriology laboratory in resource-limited countries. It is also time-consuming compared to traditionally used tools, as culture cultivation must be performed prior to DNA isolation. Therefore, more research is focused on WGS directly from clinical material. Another limitation in the implementation of WGS into routine practice is also caused by the bioinformatics processing of sequencing data. Nowadays, it requires demanding software and programming skills. However, technological advances in the coming years will lead to the development of easy-to-use online webtools such as TB-Profiler (https://tbdr.lshtm.ac.uk/, accessed on 22 October 2021) or PhyResSE (https://bioinf.fz-borstel.de/mchips/phyresse/, accessed on 22 October 2021), which are used to diagnose and determine the complete resistance profile of M. tuberculosis.

In contrast, the WGS-data are more comprehensive as these allow the precise identification of novel NTM species from human samples, prediction of genes for virulence, intracellular existence, disease, defense, and toxic compounds of NTM, thus emphasizing other applications of this method (Table 2) [58, 121, 122] . Moreover, extended datasets of clinical phenotypes and bacterial DNA sequences could resolve ambiguities in the pathogenesis of NTM. We also assume that in the future, the results of WGS will lead to wider use of personalized medicine and thus increase the effectiveness of treatment regimens. In conclusion, the more NTM genomes are sequenced, the more they will contribute to the global tracing of NTM infections and the description of important genetic determinants, thus the development of novel diagnostic tools and new therapeutic targets. 

The authors declare no conflict of interest.

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Non-tuberculous mycobacteria in lung transplant recipients: Prevalence, risk factors, and impact on survival and chronic lung allograft dysfunction

Non-tuberculous Mycobacterium infection after lung transplantation is associated with increased mortality

Advancing Translational Science for Pulmonary Nontuberculous Mycobacterial Infections. A Road Map for Research

Children with Cystic Fibrosis Are Infected with Multiple Subpopulations of Mycobacterium abscessus With Different Antimicrobial Resistance Profiles

Whole-genome analysis of mycobacteria from birds at the San Diego Zoo

Whole-Genome Sequencing Analysis to Identify Infection with Multiple Species of Nontuberculous Mycobacteria

Mycobacterium avium Complex Diversity within Lung Disease, as Revealed by Whole-Genome Sequencing

Two Rapidly Growing Mycobacterial Species Isolated from a Brain Abscess: First Whole-Genome Sequences of Mycobacterium immunogenum and Mycobacterium llatzerense

Emended description of Mycobacterium abscessus, Mycobacterium abscessus subsp. abscessus and Mycobacterium abscessus subsp. bolletii and designation of Mycobacterium abscessus subsp. massiliense comb. nov

Pulmonary disease caused by a newly identified mycobacterium: Mycolicibacterium toneyamachuris: A case report

Complete nontuberculous mycobacteria whole genomes using an optimized DNA extraction protocol for long-read sequencing

A complete high-quality MinION nanopore assembly of an extensively drug-resistant Mycobacterium tuberculosis Beijing lineage strain identifies novel variation in repetitive PE/PPE gene regions

MinION-based long-read sequencing and assembly extends the Caenorhabditis elegans reference genome

Comprehensive subspecies identification of 175 nontuberculous mycobacteria species based on 7547 genomic profiles

Intraspecies plasmid and genomic variation of Mycobacterium kubicae revealed by the complete genome sequences of two clinical isolates

Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples

Non-tuberculous mycobacteria and the rise of Mycobacterium abscessus

Non-tuberculous mycobacterial infections-A neglected and emerging problem

Mycobacterial cell wall: Structure and role in natural resistance to antibiotics

Mycobacterium abscessus: A new antibiotic nightmare

Mutations in the MAB_2299c TetR Regulator Confer Cross-Resistance to Clofazimine and Bedaquiline in Mycobacterium abscessus

The role of efflux pumps in macrolide resistance in Mycobacterium avium complex

Emergence of mmpT5 Variants during Bedaquiline Treatment of Mycobacterium intracellulare Lung Disease

New Insights in to the Intrinsic and Acquired Drug Resistance Mechanisms in Mycobacteria

Whole genome-sequencing of non-tuberculous mycobacteria

Comparative analysis of phenotypic and genotypic antibiotic susceptibility patterns in Mycobacterium avium complex

Whole-Genome Sequencing for Predicting Clarithromycin Resistance in Mycobacterium abscessus

Novel Mutations Associated with Clofazimine Resistance in Mycobacterium abscessus

Global phylogenomic analyses of Mycobacterium abscessus provide context for non cystic fibrosis infections and the evolution of antibiotic resistance

A novel DNA chromatography method to discriminate Mycobacterium abscessus subspecies and macrolide susceptibility

Whole Genome Sequencing for Clarithromycin and Amikacin Resistance Prediction and Subspecies Identification of Mycobacterium abscessus

Mycobacterium abscessus Strain Morphotype Determines Phage Susceptibility, the Repertoire of Therapeutically Useful Phages, and Phage Resistance

Nosocomial Infections Due to Nontuberculous Mycobacteria

Nosocomial outbreaks/pseudo outbreaks caused by nontuberculous mycobacteria

Nosocomial and Healthcare-Associated NTM Infections and Their Control

Whole-Genome Sequencing of the Mycobacterium tuberculosis Manila Sublineage Results in Less Clustering and Better Resolution than Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat (MIRU-VNTR) Typing and Spoligotyping

Use of bacterial whole-genome sequencing to investigate local persistence and spread in bovine tuberculosis

Whole Genome Sequencing for Determining the Source of Mycobacterium bovis Infections in Livestock Herds and Wildlife in New Zealand

Tracking and responding to an outbreak of tuberculosis using MIRU-VNTR genotyping and whole genome sequencing as epidemiological tools

Limitations of variable number of tandem repeat typing identified through whole genome sequencing of Mycobacterium avium subsp

Wholegenome sequencing and epidemiological analysis do not provide evidence for cross-transmission of Mycobacterium abscessus in a cohort of pediatric cystic fibrosis patients

The Rise of Non-Tuberculosis Mycobacterial Lung Disease. Front

Respiratory Outbreak of Mycobacterium abscessus Subspecies massiliensein a Lung Transplant and Cystic Fibrosis Center

Mycobacterium abscessus in patients with cystic fibrosis: Low impact of inter-human transmission in Italy

Population genomics of nontuberculous mycobacteria recovered from United States cystic fibrosis patients

Investigating transmission of Mycobacterium abscessus amongst children in an Australian cystic fibrosis centre

Genomic characterization of sporadic isolates of the dominant clone of Mycobacterium abscessus subspecies massiliense

Emergence and spread of a human-transmissible multidrug-resistant nontuberculous mycobacterium

The importance of early diagnosis of Mycobacterium abscessus complex in patients with cystic fibrosis

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

Epidemiology of Mycobacterium abscessus in England: An observational study

Infection Prevention and Control Guideline for Cystic Fibrosis

Contamination during production of heater-cooler units by Mycobacterium chimaera potential cause for invasive cardiovascular infections: Results of an outbreak investigation in Germany

The global outbreak of Mycobacterium chimaera infections in cardiac surgery-A systematic review of whole-genome sequencing studies and joint analysis

Global outbreak of severe Mycobacterium chimaera disease after cardiac surgery: A molecular epidemiological study

Mycobacterium chimaera in Heater-Cooler Units in Denmark Related to Isolates from the United States and United Kingdom

Mycobacterium chimaera infections after cardiovascular surgery: Lessons from a global outbreak

Genomic analysis of cardiac surgery-associated Mycobacterium chimaera infections in Italy

Mycobacterium chimaera Genomics with Regard to Epidemiological and Clinical Investigations Conducted for an Open Chest Postsurgical Mycobacterium chimaera Infection Outbreak. Open Forum Infect

Heater-cooler units: Contamination of crucial devices in cardiothoracic surgery

Healthcare-associated prosthetic heart valve, aortic vascular graft, and disseminated Mycobacterium chimaera infections subsequent to open heart surgery

Insidious Risk of Severe Mycobacterium chimaera Infection in Cardiac Surgery Patients

Mycobacterium chimaera Outbreak Associated with Heater-Cooler Devices: Piecing the Puzzle Together

Van Der Mee-Marquet, N. Cause Analysis of an Infection in Facelift Surgery Due to Mycobacterium chelonae

Mycobacterium chelonae Infection of the Buttocks Secondary to Lipofilling: A Case Report and Review of the Literature

Identification of the Infection Source of an Outbreak of Mycobacterium chelonae Keratitis After Laser in Situ Keratomileusis

Bloodstream Infections with a Novel Nontuberculous Mycobacterium Involving 52 Outpatient Oncology Clinic Patients-Arkansas

Exosome secretome and mediated signaling in breast cancer patients with nontuberculous mycobacterial disease

Laurenson, I. Investigation of two cases of Mycobacterium chelonae infection in haemato-oncology patients using whole-genome sequencing and a potential link to the hospital water supply

Two New Cases of Pulmonary Infection by Mycobacterium shigaense

Antimycobacterial Susceptibility Testing of Nontuberculous Mycobacteria

Increased Virulence of Outer Membrane Porin Mutants of Mycobacterium abscessus