**3. Results**

Table 4 below presents the percentage of strains that are sensitive, intermediate, and resistant to particular antibiotics. The classification was made on the basis of the obtained MIC values.


**Table 4.** Classification of analysed RGM species into groups: (S)-sensitive, (I)-intermediate, and (R)-resistant, based on the measured MIC values.

The data obtained in the performed antimycobacterial susceptibility test showed that amikacin and linezolid had the strongest antituberculotic activity against RGM. Most of the analysed strains were resistant to doxycycline and trimethoprim/sulfamethoxazole. The following tables (Tables 5–9) present the obtained results separately for each RGM species.

**Table 5.** Results of in vitro susceptibility testing for *M. abscessus* subsp. *abscessus* strains.



**Table 6.** Results of in vitro susceptibility testing for *M. abscessus* subsp. *massiliense* strains.

**Table 7.** Results of in vitro susceptibility testing for *M. chelonae* strains.


**Table 8.** Results of in vitro susceptibility testing for *M. mucogenicum* strains.


#### *3.1. Mycobacterium abscessus subsp. abscessus*

All strains representing *Mycobacterium abscessus* subsp. *abscessus* were sensitive only to amikacin. Of the 16 strains, 12 (75%) were also sensitive to linezolid. However, they were all resistant to minocycline, trimethoprim/sulfamethoxazole, and doxycycline. Of the 16 strains, 13 (81%) were resistant to ciprofloxacin and moxifloxacin. Among the 16 strains from this subtype, 12 (75%) were clarithromycin-resistant (MIC > 16 μg/mL) (Table 5). The GenoType NTM-DR assay confirmed the presence of the functional *erm* (41) gene in these strains, associated with inducible macrolide resistance (erm41T28 genotype). Another four strains were sensitive to clarithromycin (erm41C28 genotype).

#### *3.2. Mycobacterium abscessus subsp. massiliense*

Strains representing *Mycobacterium abscessus* subsp. *massiliense* were sensitive to linezolid (100%) and amikacin (86%). They were all resistant to trimethoprim/sulfamethoxazole, doxycycline, and ciprofloxacin. Of the 7 strains, 2 (29%) representing the above subtype were resistant to clarithromycin (Table 6). The GenoType NTM-DR assay revealed the presence of the MUT2 mutation in the *rrl* gene (constitutive resistance) (Table 1). One strain representing *Mycobacterium abscessus* subsp. *massiliense* and sensitive to clarithromycin, with the MUT1 mutation in the *rrs* gene, was resistant to amikacin (MIC > 64 μg/mL) (Table 2).

#### *3.3. Mycobacterium chelonae*

All *Mycobacterium chelonae* strains (5) were sensitive to amikacin, clarithromycin, linezolid, and tobramycin, but resistant to trimethoprim/sulfamethoxazole, ciprofloxacin, and doxycycline (Table 7).


**Table 9.** Results of in vitro susceptibility testing for *M. fortuitum* complex strains.

#### *3.4. Mycobacterium mucogenicum*

Two tested *Mycobacterium mucogenicum* strains (100%) were sensitive to clarithromycin, amikacin, cefoxitin, ciprofloxacin, moxifloxacin, and trimethoprim/sulfamethoxazole. One strain was resistant to doxycycline (MIC >16 μg/mL) (Table 8).

#### *3.5. Mycobacterium fortuitum Complex*

All strains representing *Mycobacterium fortuitum* complex (30) were sensitive to amikacin and moxifloxacin, 29 out of 30 strains were also sensitive to ciprofloxacin, 23 (77%) were resistant to clarithromycin, 17 (57%) were resistant to doxycycline, and 21 (70%) were resistant to trimethoprim/sulfamethoxazole (Table 9).

In the analysed collection of RGM, all 60 (100%) strains had low MIC values (from 0.06 to 1 μg/mL) for tigecycline, an antibiotic considered as a potential therapeutic agen<sup>t</sup> and a drug of last resort in the treatment of severe cases of mycobacteriosis.

## **4. Discussion**

With advances in molecular techniques and genetic tools, including whole genome sequencing (WGS), knowledge about the genetic diversity of NTM species and genes determining resistance to antibiotics continues to grow. Long treatment (18 to 24 months on average) and the need to use a combination of antibiotics with multiple side effects increase the importance of drug resistance testing, especially in RGM strains naturally resistant to first-line antituberculotic drugs.

Guidelines on antimicrobial susceptibility testing (AST) of atypical mycobacteria were developed by the CLSI and last updated in December 2018. Currently, CLSI M24 (3rd edition) provides recommendations on AST for slowly growing non-tuberculous mycobacteria, including *M. avium* complex (MAC), *M. kansasii*, and *M. marinum*, as well as rapidly growing mycobacteria (RGM) [34]. Since atypical mycobacteria may colonize the respiratory tract, their isolation from clinical specimens does not always correlate with the identification of an etiological factor responsible for the observed changes. This primarily refers to single sputum cultures. A negative sputum smear indicates a small number of microorganisms that are unlikely to be clinically significant, i.e., insufficient to establish a diagnosis of NTM. Therefore, detailed criteria for the diagnosis of mycobacteriosis have been developed for clinically significant isolates from the respiratory tract [27,38]. These criteria include the following:


According to the current CLSI recommendations, AST includes antimicrobial agents for RGM such as amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline (or minocycline), imipenem, linezolid, moxifloxacin, trimethoprim-sulfamethoxazole, and tobramycin (for *M*. *chelonae* only) (Table 3). Worth noting is the fact that there are insufficient data to establish MIC breakpoints for tigecycline and clofazimine, and therefore for these agents a MIC without interpretation should be given [34].

The results of AST with selected drugs may concern specific species of atypical mycobacteria. For this reason, CLSI and most experts in RGM recommend identifying RGM strains at the species or even subspecies level (Table 10), especially for the *M*. *abscessus* complex, before performing a new AST RAPMYCOI and initiating treatment [5,34,39,40].


**Table 10.** Interpretation of AST results for *M. abscessus* complex and clarithromycin.

Because the incubation period for most RGM species ranges from 2 to 5 days, the final MIC reading in the RAPMYCOI test should be performed <5 days. This is mainly due to the instability of some drugs, including carbapenems and tetracyclines. There are only two exceptions where this incubation time should be extended when performing the RAPMYCOI test. The first case concerns strains representing *M. abscessus* complex isolated from patients who had a history of long-term treatment, including patients with cystic fibrosis. Mycobacterial strains isolated from this population of patients need a longer incubation period; therefore, in some cases it may be helpful to change the incubation temperature or to establish a culture in a shaking incubator. However, if the culture incubation period is longer than 5 days, results are only reliable for AST related to two drugs: clarithromycin and amikacin. The CLSI recommends a comment on the AST report such as: this NTM strain required extended incubation and results for only clarithromycin and amikacin are reliable after incubation for >5 days (Table 10) [34].

The second exception in the RAPMYCOI test to the incubation period longer than 5 days is clarithromycin. Phenotypical detection of inducible resistance to macrolides is achieved by extending the incubation of clarithromycin to 14 days unless the MIC is ≥16 μg/mL at an earlier time point. If the clarithromycin MIC is 4 or 8 μg/mL after 14 days of incubation, the test should be repeated. If the MIC is 4 or 8 μg/mL in the retest, sequencing of the *erm* gene for the given strain is recommended.

Worth noting is the fact that several RGM species have a non-functional or absent *erm* gene and are naturally sensitive to clarithromycin [41]. Therefore, sensitivity to clarithromycin can be reported at the initial MIC reading as no prolonged incubation is required for these specific species [42].

In the presented study, we identified 60 rapidly growing mycobacterial strains and determined their antimicrobial susceptibility in accordance with CLSI guidelines.

The most frequently isolated species was *Mycobacterium fortuitum* complex, which accounted for 50% (30/60) of all identified strains. The tests demonstrated that among all RGM species this group is characterized by high sensitivity to antibiotics. Our study confirmed this thesis and showed that 100% (30/30) of the strains from this group were sensitive to amikacin and moxifloxacin, 97% (29/30) were also sensitive to ciprofloxacin, and 93% (28/30) were sensitive to linezolid. In contrast, tests with clarithromycin showed a high level of resistance for 77% (23/30) of the strains. This resistance is higher compared to that reported by Sriram et al. (100% of sensitive strains among 30 tested) and Bhalla et al. (94.1% of sensitive strains among 17 tested) [43,44].

A low rate of drug resistance according to CLSI was also found for *Mycobacterium chelonae*. In our study, 100% of the strains (5/5) were sensitive to amikacin, clarithromycin, linezolid, and tobramycin. Our findings are consistent with those reported by Bhalla et al. In the cited study, no resistance to the four above-mentioned antibiotics was found for the three tested isolates [43].

*Mycobacterium chelonae* and *Mycobacterium mucogenicum* are classified into the group of species lacking functional *erm* genes. In our study, all strains of *M. chelonae* (5/5) and *M. mucogenicum* (2/2) were sensitive to clarithromycin and no *erm* genes were detected. However, Esteban et al. detected resistance to clarithromycin associated with the presence of *erm* genes in two strains of *M. chelonae* [45]. In a study by Davalos et al., 100% (2/2) of *M. chelonae* strains were sensitive to clarithromycin. However, one strain (25%) of *M. mucogenicum* resistant to this antibiotic was detected [46]. In our study, two strains representing *M. mucogenicum* were sensitive to most of the tested antibiotics. Only one strain was resistant to doxycycline and minocycline. A different antimicrobial susceptibility profile for this species was found by Faridah et al., who reported resistance to ciprofloxacin, doxycycline, clarithromycin, and tobramycin in a strain isolated from blood [47].

Isolates representing the *Mycobacterium abscessus* complex accounted for 38% (23/60) of all identified strains and it was the second largest group. Most strains (16) represented *M. abscessus* subsp. *abscessus* subtype, while *M. abscessus* subsp. *massiliense* subtype was less frequently identified (seven strains). We did not identify *M. abscessus* subsp. *bolletii*. In our study, most strains of *M. abscessus* subsp. *abscessus* (75%) were resistant to clarithromycin (MIC >16 μg/mL). This resistance was associated with the presence of a functional *erm* gene. The situation was different for *M. abscessus* subsp. *massiliense*. Only 28% of strains representing this subtype were clarithromycin-resistant, and the MUT2 mutation in the *rrl* gene was responsible for the resistance mechanism. Our findings confirm the worldwide reports on the more frequent resistance of *M. abscessus* subsp. *abscessus* to clarithromycin compared to *M. abscessus* subsp. *massiliense* [48–50]. Considering amikacin, the vast majority of strains were sensitive to this antibiotic. Only one strain (4%) was resistant to amikacin (MIC > 64 μg/mL) and had the MUT1 mutation in the *rrs* gene. Similar findings were reported by Bhalla et al., who found 92.3% of sensitive strains [43].

Among the three tested tetracyclines (doxycycline, minocycline, and tigecycline), the lowest MICs (from 0.06 to 1 μg/mL) were found for tigecycline. If we assume the criteria for interpretation proposed by Wallace et al. (resistant strain when MIC ≥ 8 μg/mL), all tested strains (60/60) were sensitive to tigecycline [51]. Similar relationships were observed by Pang et al.: sensitivity to tigecycline was found for 96% (53/55) of strains from the *M. abscessus* complex, 91% (10/11) of *M. fortuitum* strains, and 100% (3/3) of *M. chelonae* strains [52]. Similarly, in a study by Comba et al., the MIC value was <0.25 μg/mL

for 45.7% of the strains (16/35), and from 0.25 μg/mL to 0.5 μg for 54.3% of the strains (19/35) [53]. According to worldwide reports, tigecycline is used in the treatment of the most severe infections with RGM mycobacteria, but to date there are no CLSI guidelines for the interpretation of MIC values in the AST.
