**1. Introduction**

#### **1.1. Etiology, epidemiology and possible sources of NTM infections**

The NTM group of mycobacteria is nonmotile aerobic bacilli, acid-fast (AF) staining organisms [1]. The lipid-enriched hydrophobic cell well is usually thicker than other bacteria

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

characterized by tolerance to many disinfectants, heavy metals and antibiotics [1, 2]. They are frequently found in the environment such as soil and water. They readily form biofilms, which contributes to their resistance against a variety of antibiotics [3] as well as high temperatures and a wide range of pH [4]. Environmental recovery of these NTM is the same when they do in similar culture techniques in different geographical regions [5]. However, western countries are reporting a greater prevalence of NTM infections compared to Tuberculosis (TB) than most Asian countries due to very stringent prevention and treatment of tuberculosis [6]. Not all the culture-positive samples represent infection and only half of the culture-positive patients have active respiratory infections, highlighting that NTM can be silent in presence of a normal immune response [7]. Reports suggest that older patients and women have higher chances of NTM infections [8]. As an outcome of the Human Immunodeficiency Virus (HIV) epidemic, NTM infections are frequently isolated from the blood of HIV patients [9]. In the United States, NTM cultures (more than 90%) are from pulmonary disease [10]. According to the Infectious Diseases Society of American Emerging Infections Network and Information from referral centers report, NTM infections are emerging pathogens, particularly rapidly growing mycobacteria (RGM) such as *Mycobacterium abscessus* (*M. abscessus*), *Mycobacterium chelonae* (*M. chelonae*) and *Mycobacterium fortuitum* (*M. fortuitum*) [11]. The prevalence and trend of NTM pulmonary infections are increasing, particularly in Florida and New York, calculated from United States census data from 1998 through 2005 [12]. NTM are the most common pathogens after cosmetic surgeries such as tattooing and Laser in situ keratomileusis (LASIK) [13, 14]. Increasing reports of NTM infections are expected in eye, skin, and lung due to the popularity of LASIK, increasing population of immunocompromised patients and older population. NTM pulmonary infections are found in the areas with heavy population, indicating that urban water supply increases individual's exposures to NTM [15]. NTM infections are frequently associated with farmers in Japan, suggesting that soil is the main source of infection there [16]. For NTM lung infections, aerosolization of droplets by bathroom showers may be another route of infection [17]. Water is considered to be a normal habitat for NTM and households with low water heater temperature are found to correlate with NTM infections [18]. Hospital water supply is considered to be vital in controlling NTM infections and dialysis solutions contaminations have led to the NTM outbreaks [19–21]. Contaminated tap water and increased demand of cosmetic surgeries in freestanding health centers that cannot be reviewed frequently by the infectious diseases control center are other concerns for NTM outbreaks [22].

**1.3. Laboratory diagnosis and barriers**

Culture technique is the typical standard method for the identification of suspicious NTM. The organisms must be cultured on specific media such as AF smear, Lowenstein-Jensen (LJ) media, Middlebrook media and MacConkey agar since it cannot be differentiated by Gramstain [26]. The organisms must be cultured in both liquid medium for growing a large amount of organism for other tests and solid medium to observe colony morphology and characteristic [27]. Moreover, the organisms should be further identified into subspecies level for different appropriate antimicrobial therapy. Subspecies level can be achieved by using gene sequencing, high-performance liquid chromatography (HPLC), and molecular-based methods [28]. HPLC is a fast, reliable method for identifying NTM. However, HPLC has limitations: it cannot separate between *M. abscessus* and *M. fortuitum*/*M. chelonae* [29]. Molecular probes, acridinium ester-labeled DNA probes have been made commercially and approved by the U.S. Food and Drug Administration (FDA) for the rapid identification of NTM [30]. MicroSeq 500 16S rDNA Bacterial Sequencing Kit (PE Applied Biosystems, Foster City, CA) has been developed to identify the NTM strain [31]. However, misdiagnoses frequently occur due to the low frequency of these infections, coupled with a lack of diagnostic experience for NTM infections, as well as confusing morphological features in stained smears [28]. Misdiagnosis can be complicated by incorrectly correlating laboratory results by physicians [32]. Misdiagnosis of NTM infections can lead to fatal incidents and NTM often exhibit the microbiological features of *Corynebacterium* species with long filamentous beaded appearances [33, 34]. NTM microscopic features are also similar to *Nocardia* species [35]. Therefore, clinicians are taking note of these

Nontuberculous Mycobacterial Infections: Negligent and Emerging Pathogens

http://dx.doi.org/10.5772/intechopen.80444

41

emerging infections for prompt and focused diagnosis to initiate effective treatment.

**Figure 1.** Bar graph showing the incidences of NTM in Singapore (2007–2017). Other NTM consists of *M. szulgai*, *M. terrae* complex, *M. haemophilum*, *M. intracellulare*, *M. marinum*, *M. mucogenicum*, *M. neoaurum*, *M. scrofulaceum*, *M. simiae*, *M. mageritense*, *M. wolinskyi*, *M. asiaticum*, *M. celatum*, *M. chimaera*, *M. duvalli*, *M. cookii, M. cosmeticum*, *M. chlorophenolicum*,

*M. genavense*, *M. kubicae*, *M. lentiflavum*, *M. mantenii*, *M. obuense*, *M. stomatepiae*, *M. triplex* and *M. xenopi*.

#### **1.2. Runyon's classification**

Runyon classified NTM into four groups, I–IV [23–25]. Group I, photochromogens, which usually grow slowly about 2–4 weeks and change to yellow with light exposure. Group II, scotochromogens, consist mainly of *M. gordonae* and appear as yellow colonies at 2–4 weeks in agar plates when cultured in the dark. Group III are nonphotochromogens, slowly growing mycobacteria, which grow slowly over 2–4 weeks. The rapid growers, group IV NTMs are the most pathogenic and important for human disease. They are divided into three subgroups: *M. fortuitum*, *M. chelonae/abscessus*, and *M. smegmatis*. According to the literature, they are susceptible to various antibiotics such as sulfonamide, polymyxin B, and the third- and fourth-generation fluoroquinolones [25]. Group III organisms are lung pathogens and Group IV organisms are the most important and prevalent strains for the eye, lung cutaneous and subcutaneous infections [25].

#### **1.3. Laboratory diagnosis and barriers**

characterized by tolerance to many disinfectants, heavy metals and antibiotics [1, 2]. They are frequently found in the environment such as soil and water. They readily form biofilms, which contributes to their resistance against a variety of antibiotics [3] as well as high temperatures and a wide range of pH [4]. Environmental recovery of these NTM is the same when they do in similar culture techniques in different geographical regions [5]. However, western countries are reporting a greater prevalence of NTM infections compared to Tuberculosis (TB) than most Asian countries due to very stringent prevention and treatment of tuberculosis [6]. Not all the culture-positive samples represent infection and only half of the culture-positive patients have active respiratory infections, highlighting that NTM can be silent in presence of a normal immune response [7]. Reports suggest that older patients and women have higher chances of NTM infections [8]. As an outcome of the Human Immunodeficiency Virus (HIV) epidemic, NTM infections are frequently isolated from the blood of HIV patients [9]. In the United States, NTM cultures (more than 90%) are from pulmonary disease [10]. According to the Infectious Diseases Society of American Emerging Infections Network and Information from referral centers report, NTM infections are emerging pathogens, particularly rapidly growing mycobacteria (RGM) such as *Mycobacterium abscessus* (*M. abscessus*), *Mycobacterium chelonae* (*M. chelonae*) and *Mycobacterium fortuitum* (*M. fortuitum*) [11]. The prevalence and trend of NTM pulmonary infections are increasing, particularly in Florida and New York, calculated from United States census data from 1998 through 2005 [12]. NTM are the most common pathogens after cosmetic surgeries such as tattooing and Laser in situ keratomileusis (LASIK) [13, 14]. Increasing reports of NTM infections are expected in eye, skin, and lung due to the popularity of LASIK, increasing population of immunocompromised patients and older population. NTM pulmonary infections are found in the areas with heavy population, indicating that urban water supply increases individual's exposures to NTM [15]. NTM infections are frequently associated with farmers in Japan, suggesting that soil is the main source of infection there [16]. For NTM lung infections, aerosolization of droplets by bathroom showers may be another route of infection [17]. Water is considered to be a normal habitat for NTM and households with low water heater temperature are found to correlate with NTM infections [18]. Hospital water supply is considered to be vital in controlling NTM infections and dialysis solutions contaminations have led to the NTM outbreaks [19–21]. Contaminated tap water and increased demand of cosmetic surgeries in freestanding health centers that cannot be reviewed frequently by the infectious diseases control center are other concerns for NTM outbreaks [22].

Runyon classified NTM into four groups, I–IV [23–25]. Group I, photochromogens, which usually grow slowly about 2–4 weeks and change to yellow with light exposure. Group II, scotochromogens, consist mainly of *M. gordonae* and appear as yellow colonies at 2–4 weeks in agar plates when cultured in the dark. Group III are nonphotochromogens, slowly growing mycobacteria, which grow slowly over 2–4 weeks. The rapid growers, group IV NTMs are the most pathogenic and important for human disease. They are divided into three subgroups: *M. fortuitum*, *M. chelonae/abscessus*, and *M. smegmatis*. According to the literature, they are susceptible to various antibiotics such as sulfonamide, polymyxin B, and the third- and fourth-generation fluoroquinolones [25]. Group III organisms are lung pathogens and Group IV organisms are the most important and prevalent strains for the eye, lung cutaneous and subcutaneous infections [25].

**1.2. Runyon's classification**

40 Basic Biology and Applications of Actinobacteria

Culture technique is the typical standard method for the identification of suspicious NTM. The organisms must be cultured on specific media such as AF smear, Lowenstein-Jensen (LJ) media, Middlebrook media and MacConkey agar since it cannot be differentiated by Gramstain [26]. The organisms must be cultured in both liquid medium for growing a large amount of organism for other tests and solid medium to observe colony morphology and characteristic [27]. Moreover, the organisms should be further identified into subspecies level for different appropriate antimicrobial therapy. Subspecies level can be achieved by using gene sequencing, high-performance liquid chromatography (HPLC), and molecular-based methods [28]. HPLC is a fast, reliable method for identifying NTM. However, HPLC has limitations: it cannot separate between *M. abscessus* and *M. fortuitum*/*M. chelonae* [29]. Molecular probes, acridinium ester-labeled DNA probes have been made commercially and approved by the U.S. Food and Drug Administration (FDA) for the rapid identification of NTM [30]. MicroSeq 500 16S rDNA Bacterial Sequencing Kit (PE Applied Biosystems, Foster City, CA) has been developed to identify the NTM strain [31]. However, misdiagnoses frequently occur due to the low frequency of these infections, coupled with a lack of diagnostic experience for NTM infections, as well as confusing morphological features in stained smears [28]. Misdiagnosis can be complicated by incorrectly correlating laboratory results by physicians [32]. Misdiagnosis of NTM infections can lead to fatal incidents and NTM often exhibit the microbiological features of *Corynebacterium* species with long filamentous beaded appearances [33, 34]. NTM microscopic features are also similar to *Nocardia* species [35]. Therefore, clinicians are taking note of these emerging infections for prompt and focused diagnosis to initiate effective treatment.

**Figure 1.** Bar graph showing the incidences of NTM in Singapore (2007–2017). Other NTM consists of *M. szulgai*, *M. terrae* complex, *M. haemophilum*, *M. intracellulare*, *M. marinum*, *M. mucogenicum*, *M. neoaurum*, *M. scrofulaceum*, *M. simiae*, *M. mageritense*, *M. wolinskyi*, *M. asiaticum*, *M. celatum*, *M. chimaera*, *M. duvalli*, *M. cookii, M. cosmeticum*, *M. chlorophenolicum*, *M. genavense*, *M. kubicae*, *M. lentiflavum*, *M. mantenii*, *M. obuense*, *M. stomatepiae*, *M. triplex* and *M. xenopi*.

#### **1.4. NTM incidence in Singapore (2007–2017)**

The incidences of NTM cases in Singapore are rising in the recent years, about 3000 cases per year [36] (**Figure 1**). Among NTM, *M. abscessus* is responsible for most of the identified NTM cases in Singapore, followed by *M. fortuitum, M. avium* complex and *M. chelonae* (**Figure 1**).

purulent discharge from patients with venous catheters [43, 44] and is the cause of post surgical wound infections such as liver transplant patients, electromyography and punch biopsy

Nontuberculous Mycobacterial Infections: Negligent and Emerging Pathogens

http://dx.doi.org/10.5772/intechopen.80444

43

*M. chelonae* infections are usually associated with immunocompromised hosts such as HIV patients [50]. It can be seen in postsurgical wounds and can disseminate hematogenoulsy to cause sepsis. Contaminated water is the most common source of infection and the route of

Circumscribed, red, infiltrative plaques, umbilicated papules, and pustules on the upper part of the body and face are features of *M. chelonae* skin lesions and frequently accompanied by cervical lymphadenopathy [51]. Immunocompromised patients, HIV/AIDS patients often contract *M. chelonae* infections [50]. Kidney transplant patients, liver transplant patients, tattooing, kidney dialysis patients and peritoneal dialysis patients are also frequently associated with *M. chelonae* infections [13, 52, 53]. Reports suggest that immunosuppressive drugs such as prednisolone, methotrexate, and adalimumab [54, 55], and autoimmune diseases such as Cushing's syndrome and rheumatoid arthritis are often associated with *M. chelonae* skin

The correct choice of antimicrobial agent, anatomic locations of the lesions, intracellular uptake and target binding are essential for the management of NTM cutaneous and subcutaneous infections. Moreover, an appropriate route of drug administration (oral, intravenous or intramuscular), acceptable and effective drug concentration is required for the treatment plan. Drug resistance mechanisms for rapidly growing mycobacteria (RGM) involving *erm* gene must be considered due to the prolonged treatment period. Therefore, it is critical to differentiate and identify rapidly growing mycobacterial at the subspecies level [25, 57]. The decision of choosing either surgical debridement in combination with mono or multidrug therapy, or only mono or multidrug therapy depends on the anatomical location and severity of the lesion, patient's immune status with presence of underlying pathology (**Table 1**) and the Minimum Inhibitory Concentration (MIC) breakpoints from the microbiology lab

Macrolides are the gold standard treatment for *M. abscessus* infections. They exhibit bactericidal actions against *M. abscessus* when the lesion has a small population of bacteria. Reports suggest that azithromycin and clarithromycin are the gold standard for treating *M. abscessus* infections in disseminated cases; however, there are reports suggesting the evolution of resistance against these drugs in prolonged monotherapy [11, 58]. Tigecycline, a new antibiotic,

*2.3.1. Clinical features and causes of M. chelonae cutaneous and subcutaneous infections*

procedures [44, 48, 49].

**2.3.** *Mycobacterium chelonae*

entry is direct inoculation.

infections [55, 56].

(**Tables 2** and **3**).

**2.4. NTM cutaneous and subcutaneous infections**

*2.4.1. M. abscessus cutaneous and subcutaneous infections*
