**1. Introduction**

Nontuberculous mycobacteria (NTM), also known as mycobacteria other than tuberculosis (MOTT), are ubiquitous environmental microorganisms [1]. Currently, more than 150 species are known worldwide and many of them are increasingly recognized as important human pathogens [2,3]. Based on their growth rate, NTM species are divided into slowly growing mycobacteria (SGM) and rapidly growing mycobacteria (RGM) [4]. To date, more than 75 RGM species have been identified, which represents approximately 50% of all known mycobacterial species [5]. Due to advances in molecular research, the number of newly discovered species continues to increase.

RGM species are classified into six main taxonomic groups, distinguished based on genetic relatedness and the presence of pigment. These are:


1


Tuberculosis caused by *Mycobacterium tuberculosis* complex remains a serious global health problem in developing countries [13]. However, in recent decades, the number of

**Citation:** Borek, A.; Zabost, A.; Głogowska, A.; Filipczak, D.; Augustynowicz-Kope´c, E. New RAPMYCOI SensititreTM Antimicrobial Susceptibility Test for Atypical Rapidly Growing Mycobacteria (RGM). *Diagnostics* **2022**, *12*, 1976. https://doi.org/ 10.3390/diagnostics12081976

Academic Editors: Anna Baraniak and Alessandro Russo

Received: 3 June 2022 Accepted: 12 August 2022 Published: 15 August 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

reported cases of mycobacteriosis, a disease caused by atypical mycobacteria, has increased significantly [14]. Factors contributing to the increased incidence of NTM infections include: demographic changes, ageing of the population, increased incidence of comorbidities, and immunosuppression [15]. However, the epidemiology of NTM infections remains unknown as the reporting of mycobacterial cases to public health authorities is not mandatory in most countries [16]. Undoubtedly, the diagnosis of the disease is facilitated by recently improved testing methods [17].

Epidemiological statistics indicate that people living in Asia are particularly susceptible to NTM infections. In 2014, the incidence of NTM in the Japanese population was estimated at 14.7/100,000 [18–20]. In Great Britain, the incidence of NTM infection increased from 0.9 to 2.9/100,000 between 1995 and 2006 [21]. Studies from North America and Australia revealed that the annual incidence of NTM in these regions in 1997–2010 was 3.2–9.8/100,000 [14]. In Denmark, the incidence of NTM increased between 2003 and 2008 from 0.6 to 1.5/100,000 [22]. In Poland, statistics published by the National Institute of Public Health (PZH) and the Chief Sanitary Inspectorate show that the incidence rate of mycobacteriosis was 0.69 in 2017, 0.63 in 2018, and 0.61/100,000 in 2019 [23].

Atypical mycobacteria are opportunistic pathogens, ubiquitous in the environment, and are found in fresh and marine water, soil, and on biofilms [24]. Infections mainly concern the population of high-risk patients, which includes patients with cystic fibrosis (CF), bronchiectasis, emphysema, chronic obstructive pulmonary disease (COPD), and immunoincompetence (human immunodeficiency virus (HIV) infection, organ transplant, diabetes mellitus, renal failure). Among rapidly growing mycobacteria, the highly pathogenic non-pigmented species include *M. fortuitum, M. abscessus,* and *M. chelonae*, which are responsible for more than 80% of all clinical cases [25].

The clinical manifestations of RGM infections are very diverse. They most often concern the respiratory tract, skin, soft tissues, bones and joints, lymphadenitis, or disseminated infections [26]. Chronic lung infections are usually caused by *M. abscessus* subsp. abscessus and *M. abscessus* subsp. *massiliense* [25,27]. In patients with cystic fibrosis, these pulmonary infections are associated with a very high mortality. *M. fortuitum* complex is most frequently isolated from infected skin after accidental injuries, cosmetic procedures, and laser surgery. Reportedly, *M. abscessus* is responsible for 90% of respiratory diseases caused by RGM, and *M. fortuitum* is responsible for 60–80% of postsurgical and catheterrelated infections [26]. The most common symptoms of infection caused by *M. chelonae* are diseases of the skin, bones, and soft tissues as well as ophthalmic infections, including keratitis. Rapidly growing mycobacteria are also isolated from patients with catheter-related bloodstream infection. In this case, the causative pathogens are *M. mucogenicum* and *M. fortuitum*, but also *M. neoaurum* and *M. bacteremicum* [28–31].

To determine the etiological factors of mycobacteriosis, it is necessary to correctly identify atypical mycobacteria to the species level. This is due to the different antimicrobial susceptibilities of mycobacteria. The managemen<sup>t</sup> of a wide spectrum of NTM infections is a serious challenge worldwide. The selection of the appropriate antibiotic therapy for the patient should be based on the results of in vitro antimicrobial susceptibility testing.

However, the suitability of antimicrobial susceptibility testing in the treatment of patients with mycobacteriosis remains controversial due to the discrepancy between test results and clinical response [27,32]. Good correlations demonstrated in the studies carried out to date have been shown for two groups of antibiotics: macrolides and aminoglycosides.

Macrolides (clarithromycin and azithromycin) are among the basic antibiotics used in the treatment of mycobacteriosis. All macrolides bind to the V domain in 23S rRNA on the 50S ribosome subunit [33]. Two mechanisms of resistance to this class of drugs have been identified so far among atypical mycobacteria. The first mechanism is the constitutive resistance associated with a point mutation at either the A2058 or A2059 position of the 23S rRNA (*rrl* gene). The second mechanism, defined as inducible macrolide resistance, is associated with functional *erm* genes encoding ribosomal methyltransferase. The *erm* genes have been identified in the following species: *erm* (41) in *M. abscessus* subsp.

*abscessus* (serovars I, VI, VII (80% of isolates)) and *M. abscessus* subsp. *bolletii*; *erm* (39) in *M. fortuitum*, *M*. *houstonense*, *M*. *porcinum,* and *M*. *neworleansense*; *erm* (38) in *M. smegmatis* and *M*. *goodie*; *erm* (40) in *M. mageritense* and *M. wolinskyi*. Clarithromycin-sensitive strains lack or have damaged *erm* genes. This group includes the following species: *M. abscessus* subsp. *abscessus* serovar II (Mab30), *M. abscessus* subsp. *massiliense*, *M. chelonae*, *M. immunogenum*, *M. mucogenicum group*, *M. peregrinum*, *and M. senegalense* [13,34,35].

The aminoglycosides (amikacin and tobramycin) act by binding stably to the 30S ribosomal subunit in bacterial cells, leading to misreading of the genetic code and inhibition of protein synthesis and consequently to cell death. Resistance to aminoglycosides in atypical mycobacteria is associated with single-point mutations in the 16S rRNA (*rrs* gene) [36].

According to the Clinical and Laboratory Standards Institute (CLSI), the broth microdilution method is considered the gold standard for testing the drug sensitivity of atypical RGM strains. Antimicrobial susceptibility testing should include the following antibiotics: clarithromycin, amikacin, moxifloxacin, linezolid, imipenem, cefoxitin, ciprofloxacin, doxycycline, trimethoprim/sulfamethoxazole, and tobramycin (only for *M. chelonae*). It is also recommended to determine the minimal inhibitory concentration (MIC) value for tigecycline, but to date there are no consensus breakpoints or guidelines for the interpretation of results [34,37].

There is a commercially available RAPMYCOI test for RGM from Thermo Fisher Scientific (Waltham, MA, USA) that includes all the antibiotics recommended for the treatment of RGM infections. *M. fortuitum* complex, *M. abscessus* subsp. *abscessus*, *M. abscessus* subsp. *massiliense* and *M. chelonae* are the most common rapidly growing mycobacteria (RGM) isolated in Poland.

In the presented study, the susceptibility of 60 RGM strains to 15 antibiotics was determined using the RAPMYCOI panels. The obtained results were compared with data published worldwide, which made it possible to obtain a complete picture of the drug resistance in this group of mycobacteria.

#### **2. Materials and Methods**

#### *2.1. Bacterial Strains and Growth Conditions*

The study was conducted on 60 strains of atypical mycobacteria (RGM) originally isolated from respiratory specimens (sputum, bronchial washings), in the period from 2019 to 2020 in mycobacterial laboratories in Poland.

The respiratory specimens were decontaminated with the sodium hydroxide and Nacetyl-L-cysteine (NaOH/NALC) (Chempur, Poland) method. The strains were cultured on solid media: egg-based Lowenstein-Jensen medium, Stonebrink medium, and in automated system MGIT (Becton Dickinson, Franklin Lakes, NJ, USA).

#### *2.2. Strain Identification*

For DNA extraction, the GenoLyse (Hain Lifescience, Nehren, Germany) kit was used according to protocol.

The strains were identified using the GenoType Mycobacterium CM assay ver. 2.0 (Hain Lifescience, Nehren, Germany) in accordance with the manufacturer's instructions.

*Mycobacteria* from the *Mycobacterium abscessus* complex (MABC) were identified using the GenoType NTM-DR assay (Hain Lifescience, Nehren, Germany). *M. mucogenicum* was identified using the GenoType Mycobacterium AS assay (Hain Lifescience, Nehren, Germany).

The collection of RGM strains from patients with suspected tuberculosis included the following species: *M. abscessus* subsp. *abscessus* (16), *M. abscessus* subsp. *massiliense* (7), *M. fortuitum* complex (30), *M. chelonae* (5), and *M. mucogenicum* (2).

#### *2.3. Molecular Determination of Antimicrobial Susceptibility to Macrolides and Aminoglycosides*

GenoType NTM-DR assay enabled the detection of resistance to macrolides (*erm* (41) and *rrl* genes) and aminoglycosides (*rrs* genes).

*Erm* (41) gene was only detected in members of the *M. abscessus* complex. The above test detected mutations at position 28 of the *erm* (41) gene:


In the Tables 1 and 2 below, the mutations detected by the applied test was shown.

**Table 1.** Mutations determining resistance to macrolides detected using the GenoType NTM-DR assay within the *rrl* gene.


**Table 2.** Mutations determining resistance to aminoglycosides detected using the GenoType NTM-DR assay within the *rrs* gene.


#### *2.4. Phenotypic Determination of Antimicrobial Susceptibility Profile*

Antimicrobial susceptibility was tested using broth microdilution. For this purpose, 96-well RAPMYCOI Sensititre ™ titration plates (Thermo Fisher Scientific, Waltham, MA, USA) were used, which allow for the simultaneous determination of susceptibility to 15 antibiotics.

RAPMYCOI plates contain freeze-dried antibiotics in a range of concentrations (μg/mL). The plate design and the tested antibiotic concentrations are presented in Figure 1.

At the first stage of the test, an inoculum of a mycobacterial suspension at the optical density of 0.5 McFarland scale was prepared. A total of 50 μL of inoculum was transferred to 10 mL of CAMHB medium (cation-supplemented Mueller-Hinton broth and TES buffer) (Thermo Fisher Scientific, Waltham, MA, USA). The 100 μL suspension prepared according to this protocol was pipetted onto a 96-well titration plate and incubated at 30 ◦C ± 2 ◦C. Plates with RGM were incubated for 3 to 5 days. Only for clarithromycin, the incubation period was prolonged to 14 days in order to detect inducible resistance associated with the presence of the *erm* genes. If microbial growth in the positive control sample was sufficient, MICs were measured. In cases of difficulties with visual reading, 10 μL of Alamar Blue (BIO-RAD, Hercules, CA, USA) reagen<sup>t</sup> and 25 μL of 5% Tween 80 (Fisher Scientific, Hampton, NH, USA) were added. A colour change from blue to pink indicated the growth of a strain. Measured MICs were interpreted and each strain was classified into one of three groups (sensitive (S), intermediate (I), and resistant (R)) in accordance with the CLSI guidelines (document M62, 1st edition) (37) (Table 3).


**Figure 1.** RAPMYCOI plate design: positive control (POS), amikacin (AMI), amoxicillin/clavulanic acid (AUG2), cefepime (FEP), cefoxitin (FOX), ceftriaxone (AXO), ciprofloxacin (CIP), clarithromycin (CLA), doxycycline (DOX), imipenem (IMI), linezolid (LZD), minocycline (MIN), moxifloxacin (MXF), trimethoprim/sulfamethoxazole (SXT), tigecycline (TGC), and tobramycin (TOB). The number under the antibiotic abbreviation shows its concentration in μg/mL.

**Table 3.** Antimicrobial agents and susceptibility breakpoints (MICs) for testing rapidly growing mycobacteria.

