The prevalence of nontuberculous mycobacteria (NTM)-pulmonary disease (PD) is increasing worldwide [1]. Major pathogens causing NTM-PD include the slowly growing mycobacteria (SGM) species Mycobacterium avium complex (MAC, mainly composed of M. avium and M. intracellulare) and M. kansasii, as well as the rapidly growing mycobacteria (RGM) species M. abscessus subspecies abscessus (hereafter, M. abscessus), M. abscessus subspecies massiliense (hereafter, M. massiliense), and M. fortuitum [2, 3].

Treatment guidelines for NTM-PD recommend macrolide (mainly clarithromycin)-based multidrug therapy [4, 5]. In the treatment of infections with SGM, especially MAC or M. kansasii, ethambutol and rifampicin can be used as adjunctive agents. For RGM, especially M. abscessus or M. massiliense, parenteral antibiotics, including amikacin, imipenem, and tigecycline, should be used for several months. RGM are usually highly drug-resistant and few drugs are effective. Clarithromycin is a cornerstone agent for NTM-PD treatment, while amikacin is crucial as a parenteral agent for NTM-PD and as salvage therapy for refractory NTD-PD caused by MAC.

Benzimidazole, a heterocyclic compound with imidazole and benzene rings, has gained attention for its biological properties in infectious disease treatment. [6]. Several benzimidazole derivatives have shown efficacy against M. tuberculosis, and their pharmaco-toxicological properties have been reported [7, 8]. The benzimidazole derivative SPR719 (formerly known as VXc-486) blocks mycobacterial gyrase ATPase and exhibits in vitro activity against some NTMs [9], but its effect on clinical NTM isolates or strains resistant to major antibiotics is unknown. We evaluated the in vitro activity of SPR719 against NTM clinical isolates, including clarithromycin- and amikacin-resistant isolates, from patients with NTM-PD.

We obtained 325 clinical NTM isolates from patients newly diagnosed as having NTM-PD at Samsung Medical Center, Seoul, Korea between 2010 and 2018 who did not have a history of antibiotic treatment for NTM-PD. These isolates belonged to six major species and/or taxonomic groups of pathogenic NTM (63 M. avium, 58 M. intracellulare, 58 M. kansasii, 65 M. abscessus, 67 M. massiliense, and 14 M. fortuitum). We also examined a separate set of 57 clarithromycin-resistant isolates (10 M. avium, 17 M. intracellulare, 13 M. abscessus, and 17 M. massiliense) and 44 amikacin-resistant isolates (12 M. avium, 17 M. intracellulare, 9 M. abscessus, and 6 M. massiliense), which were confirmed to have rrl (encoding 23S rRNA) and rrs (encoding 16S rRNA) mutations related to clarithromycin and amikacin resistance, respectively [10]. These drug-resistant isolates were obtained from patients who had previously been diagnosed as having NTM-PD and were treated with antibiotics or identified as having refractory NTM-PD, including infections caused by NTM strains reported in our previous studies [11^-15]. To confirm clarithromycin and amikacin resistance in the clinical strains, we performed two susceptibility testing procedures. Initially, the Korean Institute of Tuberculosis (Seoul, South Korea) conducted susceptibility testing using the broth microdilution method for patient care diagnosis. Subsequently, separate susceptibility testing of SPR719 was conducted on clinical isolates in our laboratory. The isolates were recultured to obtain a single colony, and strain DNA was extracted. Finally, gene (16S rRNA, ropB, hsp65) sequencing was used for identification, and the isolates were preserved for research purposes. All data were obtained from an Institutional Review Board-approved observational cohort study performed at Samsung Medical Center (ClinicalTrials.gov identifier: NCT00970801).

In vitro susceptibility testing of SPR719 was performed using the broth microdilution method according to the CLSI guidelines [16, 17]. Cation-adjusted Mueller–Hinton broth (CAMHB) (Difco Laboratories, Detroit, MI, USA) was used for RGM; CAMHB with 5% oleic–albumin–dextrose–catalase (CAMHB-OADC) was used for SGM. SPR719 dissolved in dimethyl sulfoxide was serial-diluted and dispensed into CAMHB or CAMHB-OADC in 96-well plates; a 0.5-McFarland standard bacterial suspension was diluted 1:200 and inoculated in the same way.

The minimum inhibitory concentration (MIC) was determined after incubation in a 36°C, 75%–80% humidity incubator; RGM isolates were incubated for 3–5 days, whereas SGM isolates were incubated for >10 days. After MIC measurement, aliquots above the MIC were taken from the 96-well plates and inoculated on 7H10 agar without SPR719, followed by incubation of RGM (3–4 days) and SGM (≥10 days). After incubation, the minimum bactericidal concentration (MBC) was determined, using M. peregrinum ATCC 700686, M. abscessus ATCC 19977, M. avium ATCC 700898, and M. kansasii ATCC 12478 as controls.

The in vitro activity of SPR719 against the 325 treatment-naive NTM isolates is summarized in Table 1, including the MIC range, minimum concentration required to inhibit 50% of the bacteria (MIC₅₀), minimum concentration to inhibit 90% of bacteria (MIC₉₀), MBC range, minimum concentration to kill 50% of the bacteria (MBC₅₀), and minimum concentration to kill 90% of the bacteria (MBC₉₀) analyzed according to NTM species, including SGM (M. avium, M. intracellulare, and M. kansasii) and RGM (M. abscessus, M. massiliense, and M. fortuitum) (Table 1).

Table 1 . MIC and MBC data for SPR719 against treatment-naive clinical NTM isolates (N=325)

NTM species	N of isolates with indicated MIC (µg/mL)											MIC range (µg/mL)	MIC50 (µg/mL)	MIC90 (µg/mL)
	0.031	0.062	0.125	0.25	0.5	1	2	4	8	16	>16
Mycobacterium avium (N=63)			1 (2)			10 (16)	33 (52)	14 (22)		2 (3)	3 (5)	0.125−16	2	4
M. intracellulare (N=58)				1 (2)	11 (19)	32 (55)	5 (9)	7 (12)	2 (3)			0.25−8	1	4
M. kansasii (N=58)	16 (28)	30 (52)	4 (7)	2 (3)	2 (3)	1 (2)		2 (3)			1 (2)	0.031−16	≤0.062	0.25
M. abscessus (N=65)*			1 (2)		2 (3)	15 (23)	29 (44)	16 (25)	2 (3)			0.125−8	2	4
M. massiliense (N=67)		6 (9)		10 (15)	17 (25)	21 (31)	10 (15)	3 (5)				0.062−4	1	2
M. fortuitum (N=14)				1 (7)	1 (7)	4 (29)	3 (21)	3 (21)	2 (15)			0.25−8	2	8
	N of isolates with indicated MBC (µg/mL)											MBC range (µg/mL)	MBC50 (µg/mL)	MBC90 (µg/mL)
	0.031	0.062	0.125	0.25	0.5	1	2	4	8	16	>16
M. avium (N=63)			1 (2)			8 (13)	32 (50)	11 (18)	3 (5)	4 (6)	4 (6)	0.125−16	2	16
M. intracellulare (N=58)				1 (2)	3 (5)	18 (31)	23 (39)	5 (9)	3 (5)	5 (9)		0.25−16	2	8
M. kansasii (N=58)	11 (19)	24 (41)	12 (20)	4 (7)	3 (5)	1 (2)			1 (2)	1 (2)	1 (2)	0.031−16	0.062	0.5
M. abscessus (N=65)*					1 (2)	4 (6)	19 (29)	31 (47)	9 (14)	1 (2)		0.5−16	4	8
M. massiliense (N=67)		1 (2)	1 (2)		3 (4)	16 (23)	30 (45)	15 (22)	1 (2)			0.062−8	2	4
M. fortuitum (N=14)					2 (14)	2 (14)	4 (29)	1 (7)	5 (36)			0.5−8	2	8

Data are presented as number (%). *42 of 65 M. abscessus isolates showed inducible resistance in phenotypic drug susceptibility testing.

Abbreviations: MIC, minimum inhibitory concentration; MIC₅₀, minimum concentration required to inhibit 50% of bacteria; MIC₉₀, minimum concentration to inhibit 90% of bacteria; MBC, minimum bactericidal concentration; MBC₅₀, minimum concentration required to kill 50% of bacteria; MBC₉₀, minimum concentration required to kill 90% of bacteria; NTM, nontuberculous mycobacteria.

The MIC of SPR719 was ≤2 µg/mL in 70% of clinical M. avium isolates. Both the MIC and MBC ranged from 0.125 to 16 µg/mL. The MIC₉₀ and MBC₉₀ were 4 and 16 µg/mL, respectively, which were comparable to the values obtained with M. intracellulare. The MIC of SPR719 was ≤1 µg/mL for most of the M. kansasii isolates (95%). The MIC and MBC of SPR719 for M. kansasii ranged from 0.031 to 16 µg/mL; the MIC₉₀ and MBC₉₀ were 0.25 and 0.5 µg/mL, respectively, and thus much lower than the values determined for the MAC isolates.

The MIC of SPR719 was ≤2 µg/mL in 72% of clinical M. abscessus isolates. The MIC and MBC ranges were 0.125–8 and 0.5–16 µg/mL, and the MIC₉₀ and MBC₉₀ were 4 and 8 µg/mL, respectively. The MIC of SPR719 for most M. massiliense isolates (95%) was also ≤2 µg/mL; the MIC₉₀ and MBC₉₀ were 2 and 4 µg/mL, respectively, and thus lower than the respective values against the M. abscessus isolates. The MIC of SPR719 for 64% of the M. fortuitum isolates was ≤2 µg/mL. The MIC range, MBC range, MIC₉₀, and MBC₉₀ were comparable to the values determined for M. abscessus.

The in vitro activity results of SPR719 against clarithromycin-resistant SGM and RGM isolates (N=57) are shown in Table 2. The MIC of SPR719 was ≤2 µg/mL for 80% of clarithromycin-resistant M. avium isolates. The MIC and MBC of SPR719 ranged from 0.5 to 8 µg/mL; both the MIC₉₀ and MBC₉₀ were 8 µg/mL. These values were roughly comparable to those determined for M. intracellulare. The MIC of SPR719 was also ≤2 µg/mL for 85% of the clinical isolates of clarithromycin-resistant M. abscessus. The MIC and MBC ranges were 0.062–4 and 2–8 µg/mL, and the MIC₉₀ and MBC₉₀ were 4 and 8 µg/mL, respectively. The MIC of SPR719 was ≤2 µg/mL for 88% of clarithromycin-resistant M. massiliense isolates, and the MIC range and MIC₉₀ were the same as determined in M. abscessus. The MBC range and MBC₉₀ were 0.25–4 and 4 µg/mL, respectively, and thus slightly lower than the values determined in M. abscessus (Table 2).

Table 2 . MIC and MBC data for SPR719 against clarithromycin-resistant and amikacin-resistant clinical NTM isolates

NTM species	N of isolates with indicated MIC (µg/mL)											MIC range (µg/mL)	MIC50 (µg/mL)	MIC90 (µg/mL)
	0.031	0.062	0.125	0.25	0.5	1	2	4	8	16	>16
Clarithromycin-resistant isolates (N=57)
Mycobacterium avium (N=10)					3 (30)	2 (20)	3 (30)	1 (10)	1 (10)			0.5–8	1	8
M. intracellulare (N=17)		1 (6)			3 (18)	6 (35)	3 (17)	2 (12)	2 (12)			0.062–8	1	8
M. abscessus (N=13)		1 (8)			2 (15)	6 (47)	2 (15)	2 (15)				0.062–4	1	4
M. massiliense (N=17)		2 (12)	1 (6)	4 (23)	4 (23)	2 (12)	2 (12)	2 (12)				0.062–4	0.5	4
Amikacin-resistant isolates (N=44)
M. avium (N=12)						7 (59)	4 (33)		1 (8)			1–8	1	2
M. intracellulare (N=17)					4 (24)	6 (35)	5 (29)	2 (12)				0.5–4	1	4
M. abscessus (N=9)						1 (11)	7 (78)	1 (11)				1–4	2	4
M. massiliense (N=6)					3 (50)	2 (33)	1 (17)					0.5–2	0.5	2
	N of isolates with indicated MBC (µg/mL)											MBC range (µg/mL)	MBC50 (µg/mL)	MBC90 (µg/mL)
	0.031	0.062	0.125	0.25	0.5	1	2	4	8	16	>16
Clarithromycin-resistant isolates (N=57)
M. avium (N=10)					2 (20)	2 (20)	4 (40)	1 (10)	1 (10)			0.5–8	2	8
M. intracellulare (N=17)				1 (6)	2 (12)	5 (29)	5 (29)	2 (12)	1 (6)	1 (6)		0.25–16	1	8
M. abscessus (N=13)							3 (23)	6 (46)	4 (31)			2–8	4	8
M. massiliense (N=17)				4 (23)	2 (12)	3 (18)	3 (18)	5 (29)				0.25–4	1	4
Amikacin-resistant isolates (N=44)
M. avium (N=12)						6 (50)	4 (34)	1 (8)	1 (8)			1–8	1	4
M. intracellulare (N=17)					1 (6)	6 (35)	7 (41)	1 (6)	2 (12)			0.5–8	2	8
M. abscessus (N=9)							1 (11)	7 (78)	1 (11)			2–8	4	8
M. massiliense (N=6)						4 (67)	2 (33)					1–2	1	2

Data are presented as number (%).

Abbreviations: MIC, minimum inhibitory concentration; MIC₅₀, minimum concentration required to inhibit 50% of bacteria; MIC₉₀, minimum concentration to inhibit 90% of bacteria; MBC, minimum bactericidal concentration; MBC₅₀, minimum concentration required to kill 50% of bacteria; MBC₉₀, minimum concentration required to kill 90% of bacteria; NTM, nontuberculous mycobacteria.

The SPR719 in vitro activity against 44 amikacin-resistant SGM and RGM strains is also shown in Table 2. The MIC of SPR719 was ≤2 µg/mL for 92% of the amikacin-resistant M. avium isolates. Both the MIC and MBC ranged from 1 to 8 µg/mL. The MIC₉₀ and MBC₉₀ were 2 and 4 µg/mL, respectively, which were roughly comparable to the values determined for M. intracellulare. The MIC of SPR719 was ≤2 µg/mL for 89% of the clinical amikacin-resistant M. abscessus isolates. The MIC and MBC ranges were 1–4 and 2–8 µg/mL, and the MIC₉₀ and MBC₉₀ were 4 and 8 µg/mL, respectively. The MIC of SPR719 was ≤2 µg/mL for all amikacin-resistant M. massiliense isolates. The MIC and MBC ranges were 0.5–2 and 1–2 µg/mL, respectively, and both the MIC₉₀ and MBC₉₀ were 2 µg/mL, which was slightly lower than the values determined for M. abscessus.

Overall, our results show that the MIC and MBC of SPR719 are similar for the major pathogenic NTM species, including clarithromycin- and amikacin-resistant clinical isolates from patients newly diagnosed as having NTM-PD without prior antibiotic exposure. The exception was M. kansasii, as the MIC and MBC values of SPR719 were lower for these isolates than for other species and were quite similar among isolates within the species, demonstrating the bactericidal properties of SPR719. The MIC₉₀ and MBC₉₀ of SPR719 against M. kansasii were very low (8- to 32-fold lower than those obtained with the other tested NTM species, respectively), indicating the potential of SPR719 as a new therapeutic agent for NTM-PD caused by M. kansasii. There was also little difference between the MIC and MBC values of SPR719 against the other tested NTM species, including clarithromycin- and amikacin-resistant strains. The comparable in vitro activity of SPR719 for SGM and RGM, regardless of drug-resistance status, suggests that this drug could be used in the treatment of NTM-PD caused by several different NTM species. However, additional research focusing on clinical applications is crucial as our study was solely based on in vitro results.

Most M. abscessus strains have inducible resistance to macrolide antibiotics due to a specific sequence in the erm41 gene region, resulting in high-level resistance after 14 days, according to in vitro activity measurements [10]. Although most (65%, 42/65) of the treatment-naïve M. abscessus strains exhibited clarithromycin-induced resistance, the lack of a significant difference between the SPR719 MIC and MBC values suggests the possibility of some bactericidal effect. However, it is necessary to evaluate the efficacy of SPR719 by analyzing a large number of M. abscessus strains with induced resistance to clarithromycin.

Our results are in line with a recent study showing the effectiveness of SPR719 against NTM in vitro [18]. However, we analyzed only a few important SGM and RGM strains and not many other strains. Another study reported the in vitro activity of SPR719 against other NTM species such as M. ulcerans, M. marinum, and M. chimaera, with MIC values ranging from 0.125 to 4 µg/mL [19]. A first-in-human phase 1 study of SPR720 (a phosphate prodrug of SPR719) was conducted in 2021, which evaluated the safety, tolerability, and pharmacokinetics of SPR720/SPR719 [20]. The results demonstrated that once-daily dosing of SPR720 was well-tolerated and had acceptable pharmacokinetics, supporting its use to treat NTM-PD. Our results and those of other studies thus support the use of SPR719 as a therapeutic agent in the treatment of NTM-PD, especially for infections caused by M. kansasii, although further confirmatory testing is warranted.

None.

Kim DH, Kim SY, and Jhun BW contributed to the study conception and design; Kim DH and Kim SY were responsible for conducting the experiments; Kim DH, Zo S, and Jhun BW drafted the manuscript; and Jhun BW supervised the study. All authors read and approved the final manuscript.

None declared.

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MSIT) (NRF-2019R1F1A1056568) and by the Basic Science Research Program through the NRF, funded by the Ministry of Education (NRF-2020R1I1A1A01066970 to DHK).