Resistance Patterns of Mycobacterium tuberculosis Complex Clinical Isolates to Bedaquiline, Delamanid, and Linezolid in Southern Türkiye

2.1. Phenotypic Drug Susceptibility Testing (DST) and Genotypic Analyses Were Performed for BDQ, DLM and LZD

2.1.1. Bedaquiline MIC Distribution

BDQ MICs ranged from 0.0125 to 0.8 µg/mL among fully drug-susceptible strains and 0.2 and 6.4 µg/mL among MDR-TB isolates. The H37Rv showed an MIC of 0.4 µg/mL (Figure 1). Using the WHO critical concentration (CC) of 1 µg/mL for MGIT [16], 66/71 (93.0%) isolates were classified as susceptible and 5/71 (7.0%) as resistant. All BDQ-resistant isolates were MDR-TB 5/41 (12.2%, 95% CI: 4.1–26.2%) (Table 1); 80% of the isolates were INH+RIF+PZA resistant, and the remaining 20% were INH+RIF+PZA+EMB resistant.

For BDQ, according to the EUCAST (European Committee for Antimicrobial Susceptibility Testing) clinical breakpoint (CB) of 0.25 µg/mL [16], 22/71 (31.0%) isolates were phenotypic wild-type (pWT) with MIC < 0.25 μg/ml, while 49/71 (69.0%) were non-wild-type with MIC ≥ 0.25 μg/ml. Among the non-wild-type isolates, 77.6% (38/49) were MDR-TB and 22.4% (11/49) were fully drug susceptible. BDQ MIC was significantly higher (p < 0.001) in MDR-TB isolates (0.50 µg/mL [IQR, 0.40–0.80]) compared to drug-susceptible isolates (0.20 µg/mL [IQR, 0.20–0.40]) with a higher MIC90 (2.0 vs 0.4 µg/mL) (Table 1).

2.1.2. Delamanid MIC Distribution

Delamanid MIC Distribution for Delamanid, MICs for fully drug-susceptible strains ranged from 0.005 to 0.06 µg/mL, whereas for MDR-TB isolates, MIC values ranged from 0.02 to 0.32 µg/mL (Figure 1). The H37Rv reference strain exhibited a MIC of 0.01 µg/mL. Using the WHO critical concentration (CC) of 0.06 µg/mL [16], 68/71 (95.8%) isolates were classified as susceptible, whereas 3/71 (4.2%) were classified as resistant. Of the 3 resistant isolates, 2/3 (66.7%) were MDR-TB, with 2/41 (4.9%, 95% CI: 0.6–16.5%) resistant (Table 1).; among these, 50% were INH+RIF+PZA, and 50% were INH+RIF+PZA+STR resistant. One-third (33.3%) of the DLM-resistant isolates were fully drug-susceptible strains (3.3%, 95% CI: 0.1–17.2%). The median MIC for delamanid was 0.01 µg/mL (IQR, 0.005–0.01) for drug-susceptible and 0.02 µg/mL (IQR, 0.02–0.05) for MDR-TB isolates. The MIC₉₀ was also higher in MDR-TB (0.05 µg/mL) than drug-susceptible (0.04 µg/mL) isolates, but the difference was statistically significant (p = 0.012) (Table 1).

2.1.3. Linezolid MIC Distribution

The MICs for LZD were 0.125 to 0.5 µg/mL for fully drug-susceptible strains and 0.125 to 2 µg/mL for MDR-TB strains (Figure 1). The MIC of the H37Rv strain was 0.5 µg/mL. Using the WHO critical concentration (CC) of 1 µg/mL [16], 66 of 71 (93.0%) strains were susceptible and 5 of 71 (7.0%) strains were resistant. All resistant isolates were resistant to INH and RIF (12.2%, 95% CI: 4.1–26.2) (Table 1), and 80% were also resistant to PZA. For drug-susceptible isolates, the median MIC for linezolid was 0.4 µg/mL (IQR, 0.25–0.50), and for MDR-TB isolates, it was 0.50 µg/mL (IQR, 0.25–0.50). The MIC 90 values were unchanged at 0.5 µg/mL in both groups. The difference was not significant statistically (p = 0.089) (Table 1).

Figure 1. Heatmap of MGIT-based MIC distributions among fully drug-susceptible and multidrug-resistant tuberculosis (MDR-TB) Mycobacterium tuberculosis complex isolates. (A) MIC distributions among fully drug-susceptible MTBC isolates (n = 30); (B) MIC distributions among MDR-TB MTBC isolates (n = 41); and (C) drug concentration dilution series used for MIC determination for bedaquiline, delamanid, and linezolid. Drug concentrations are expressed in µg/mL.

Figure 1. Heatmap of MGIT-based MIC distributions among fully drug-susceptible and multidrug-resistant tuberculosis (MDR-TB) Mycobacterium tuberculosis complex isolates. (A) MIC distributions among fully drug-susceptible MTBC isolates (n = 30); (B) MIC distributions among MDR-TB MTBC isolates (n = 41); and (C) drug concentration dilution series used for MIC determination for bedaquiline, delamanid, and linezolid. Drug concentrations are expressed in µg/mL.

Table 1. Comparative MIC distributions, resistance rates, and statistical analysis of bedaquiline, delamanid, and linezolid among Fully drug -susceptible and MDR-TB isolates.

Antibiotic	MTBC isolates (n=71)	MIC₅₀ (µg/mL)	IQR (µg/mL)	MIC₉₀ (µg/mL)	Resistant n (%) [95% CI]	p-value (MIC)
Bedaquiline	Fully drug -susceptible (n=30)	0.2	0.2–0.4	0.4	0/30 (0%) [0–11.5]	<0.001
	MDR-TB (n=41)	0.5	0.4–0.8	2	5/41 (12.2%) [4.1–26.2]
Delamanid	Fully drug -susceptible (n=30)	0.01	0.005–0.01	0.04	1/30 (3.3%) [0.1–17.2]	0.012
	MDR-TB (n=41)	0.02	0.02–0.05	0.05	2/41 (4.9%) [0.6–16.5]
Linezolid	Fully drug -susceptible (n=30)	0.4	0.25–0.5	0.5	0/30 (0%) [0–11.5]	0.089
	MDR-TB (n=41)	0.5	0.25–0.5	0.5	5/41 (12.2%) [4.1–26.2]

IQR, interquartile range; CI, confidence interval. MIC values are expressed as median (MIC₅₀), IQR, and MIC₉₀. MIC₅₀ represents the median MIC; MIC₉₀ represents the concentration inhibiting 90% of isolates Resistance was defined according to WHO critical concentrations (bedaquiline: 1 µg/mL; delamanid: 0.06 µg/mL; linezolid: 1 µg/mL). P values for MIC distributions were calculated using the Mann–Whitney U test.

Table 1. Comparative MIC distributions, resistance rates, and statistical analysis of bedaquiline, delamanid, and linezolid among Fully drug -susceptible and MDR-TB isolates.

Antibiotic	MTBC isolates (n=71)	MIC₅₀ (µg/mL)	IQR (µg/mL)	MIC₉₀ (µg/mL)	Resistant n (%) [95% CI]	p-value (MIC)
Bedaquiline	Fully drug -susceptible (n=30)	0.2	0.2–0.4	0.4	0/30 (0%) [0–11.5]	<0.001
	MDR-TB (n=41)	0.5	0.4–0.8	2	5/41 (12.2%) [4.1–26.2]
Delamanid	Fully drug -susceptible (n=30)	0.01	0.005–0.01	0.04	1/30 (3.3%) [0.1–17.2]	0.012
	MDR-TB (n=41)	0.02	0.02–0.05	0.05	2/41 (4.9%) [0.6–16.5]
Linezolid	Fully drug -susceptible (n=30)	0.4	0.25–0.5	0.5	0/30 (0%) [0–11.5]	0.089
	MDR-TB (n=41)	0.5	0.25–0.5	0.5	5/41 (12.2%) [4.1–26.2]

IQR, interquartile range; CI, confidence interval. MIC values are expressed as median (MIC₅₀), IQR, and MIC₉₀. MIC₅₀ represents the median MIC; MIC₉₀ represents the concentration inhibiting 90% of isolates Resistance was defined according to WHO critical concentrations (bedaquiline: 1 µg/mL; delamanid: 0.06 µg/mL; linezolid: 1 µg/mL). P values for MIC distributions were calculated using the Mann–Whitney U test.

2.2. Genotypic Analysis of Drug Resistance-Associated Mutations

The atpE gene mutations were identified in nine of 71 (12.7%) isolates. All the mutant strains were multidrug-resistant TB, and no mutations have been identified in fully drug-susceptible isolates. All mutant isolates were from BDQ-naive patients and had MIC values >0.25 µg/mL (MIC range 0.5–6.4 µg/mL). Of these, 55.6% were phenotypically resistant and 44.4% susceptible. Sequence analysis of the 244-bp atpE gene revealed eight SNVs, but no insertions or deletions. The most frequent mutation identified was G73A (G25S) in five isolates, followed by C60G (G20G) and G61C (A21P). Additional variations were found in separate isolates G70C(A24P), C72G(A24P), A64C(I22L), C66G(I22M), and C228G(F76L). Four isolates presented complex mutation patterns with the following combined substitutions: G73A/C60G/G61C, G70C/C72G, C66G/G73A, and G61C/G73A. No mutations were detected in the Rv0678 gene (Table 2).

Regarding DLM genetic resistance, one fully drug-susceptible isolate harbored a single G242A (Gly81Asp) mutation in the ddn gene but remained phenotypically susceptible (MIC 0.04 µg/mL). No mutation was detected in the fbiA gene, rplC gene, and rrl gene in the 71 isolates for DLM and LZD, respectively (Table 2).

4.1. Selection, Culture, Identification, and Drug Susceptibility Testing of MTBC Clinical Isolates

This study included a total of 71 MTBC isolates isolated from clinical samples of TB suspects sent to Cukurova University Tropical Disease Research and Application Center, Ministry of Health, Public Health Institution; and Adana Regional Tuberculosis Laboratory (THAUM), Türkiye, between January 2021 and January 2023; duplicate specimens were excluded. Only one isolate per patient was analyzed. Clinical specimens were processed according to the routine THAUM laboratory. Mycobacterial cultures were performed using the BACTEC MGIT 960 system (Becton Dickinson, Sparks, MD, USA). When an MGIT tube was flagged positive, a smear was prepared and examined by Ziehl–Neelsen staining to confirm the presence of acid-fast bacilli (AFB). Positive cultures were subsequently subjected to Mycobacterial identification. Where required, positive MGIT cultures were subcultured onto Löwenstein–Jensen (LJ) medium and incubated at 37 ± 1 °C for further confirmation and characterization.

All isolates included in the study were confirmed as MTBC by MPT64 antigen testing, with molecular analysis providing additional confirmation through detection of Mycobacterium- and MTBC-associated targets. A positive MPT64 antigen-based immunochromatographic card test was used for initial confirmation of MTBC. In parallel, genomic DNA was extracted from all 71 isolates using the Mickle mechanical disruption method. Molecular identification was performed using two PCR assays. Each PCR was conducted in a final reaction volume of 25 μL containing 12.5 μL of master mix, 0.25 μL each of forward and reverse primers, 6 μL of nuclease-free water, 1 μL of dimethyl sulfoxide (DMSO), and 5 μL of genomic DNA. Amplification was performed with an initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 63°C for 1 min, and extension at 72°C for 1 min, with a final extension at 72°C for 7 min. PCR products were resolved by agarose gel electrophoresis and visualized for the presence of the expected amplicons.

The first PCR targeted a specific region of the hsp65 gene using primers TB11 and TB12 for confirmation of Mycobacterium spp. A 441-bp amplicon was considered indicative of a Mycobacterium-positive isolate. Isolates positive for Mycobacterium spp. were subsequently tested by PCR targeting the IS6110 region using primers INS1 and INS2 for molecular confirmation of the Mycobacterium tuberculosis complex (MTBC). The presence of the expected 245-bp amplicon was considered positive for MTBC. Primer sequences, target regions, and expected amplicon sizes are provided in Supplementary File S1. Isolates lacking the IS6110 amplicon were not classified as MTBC. All isolates included in the present study were confirmed as MTBC by concordant MPT64 antigen detection and molecular identification based on Mycobacterium- and MTBC-associated targets. Species-level differentiation within the MTBC was not performed. Phenotypic drug susceptibility testing (DST) was carried out for streptomycin (STR), isoniazid (INH), rifampicin (RIF), ethambutol (EMB), and pyrazinamide (PZA) using the BACTEC MGIT960. Of the isolates, 30 were fully drug-susceptible MTBC isolates, and 41 were MDR-TB. M. tuberculosis H37Rv was utilized as a control in all phenotypic and genotypic analyses.

4.2. Drug Susceptibility Testing (DST)

As pure powders were unavailable, commercial tablet formulations of bedaquiline (SIRTURO®, 100 mg; Janssen, Belgium), delamanid (Deltyba®, 50 mg; Otsuka, Japan), and linezolid (600 mg; Koçak, Türkiye) were used. According to the manufacturers’ product information, each tablet contained the active ingredient and the corresponding excipients and was finely powdered and dissolved in the appropriate solvent. Commercial tablet formulations may affect the results due to the presence of excipients; however, a standardized preparation minimizes this variability, and reliable susceptibility results were reported.

4.3. Testing for Phenotypic Drug Susceptibility and MIC

Quantitative DST was performed using the semi-automated MGIT 960 system with EpiCenter software and the TB eXiST module according to the WHO Critical Drug Concentrations Guidelines [12,13]. Because analytical-grade drug powders were not available, bedaquiline (BDQ), delamanid (DLM), and linezolid (LZD) were prepared from commercially available pharmaceutical formulations. The tablets were finely powdered before preparation of the stock solutions. Stock solutions were prepared based on the labeled active ingredient content and subsequently diluted to obtain the working solutions required for susceptibility testing. All prepared solutions were aliquoted and stored at −70°C until use.

Drug concentrations were calculated from the labeled active ingredient content and prepared using a standardized approximately 84-fold dilution factor to achieve the desired final concentrations in the MGIT tubes. For BDQ, 100 mg of labeled active BDQ (equivalent to 100 mg BDQ base according to the manufacturer's product information) was dissolved in 25 mL of sterile DMSO to obtain a stock solution at 4,000 µg/mL. This solution was subsequently diluted with DMSO to 800 µg/mL and then to a working concentration of 84 µg/mL [12]. BDQ was tested over a final concentration range of 0.0125–6.4 µg/mL.

For DLM, 50 mg of labeled active DLM (equivalent to 50 mg of DLM base according to the manufacturer's product information) was dissolved in 12.5 mL of sterile DMSO to obtain a 1,000 µg/mL stock solution. The stock solution was diluted 1:10 with sterile distilled water to obtain a 100 µg/mL intermediate solution, which was subsequently diluted to a working concentration of 5 µg/mL [12]. DLM was tested at final concentrations ranging from 0.0025 to 0.32 µg/mL.

For LZD, 600 mg of LZD fumarate salt (equivalent to 600 mg of LZD base according to the manufacturer's product information) was dissolved in 600 mL of sterile DMSO to obtain a 1,000 µg/mL solution (Solution A). Subsequently, 4 mL of Solution A was diluted to a final volume of 5 mL with SDW, yielding an 800 µg/mL solution (Solution B). Then, 1.05 mL of Solution B was added to 8.95 mL of SDW to obtain the 84 µg/mL working solution [12]. LZD was tested at final concentrations ranging from 0.0625 to 2 µg/mL.

For DST, 0.8 mL of oleic acid–albumin–dextrose–catalase (OADC) supplement, 0.1 mL of the appropriate drug solution, and 0.5 mL of bacterial suspension were added to 7.0 mL of MGIT medium, resulting in a total volume of approximately 8.4 mL. Under these conditions, the final DMSO concentration was approximately 2.4% (v/v), and the drug solutions were diluted approximately 84-fold in the MGIT tubes. Drug-free growth-control tubes were prepared by diluting the bacterial suspension 1:100 in sterile saline and inoculating 0.5 mL of the diluted suspension into MGIT tubes.

MGIT tubes were incubated at 37°C and monitored using the MGIT 960 system. Testing was considered valid when the growth-control tube reached a growth unit (GU) value of 400 within 28 days. For drug-containing tubes, a GU value ≥100 was interpreted as resistant, whereas a GU value <100 was interpreted as susceptible [13]. Phenotypic susceptibility was determined according to the WHO critical concentrations [12], and the distribution of MICs was analyzed with EUCAST the epidemiological cutoff (ECOFF) value is the MIC that identifies the top limit for the wild-type population [13].

4.4. Genotypic Drug Susceptibility Testing and DNA Sequencing

Genotypic data were obtained from genomic DNA extracted from 71 MTBC isolates using the Mickle mechanical disruption method. DNA was quantified and stored at −20°C

Until analysis.PCR was used to amplify resistance-associated genes: atpE (Rv1305) and Rv0678 for BDQ; ddn (Rv3547) and fbiA (Rv3261) for DLM; and rrl (23S rRNA) and rplC for LZD, following WHO molecular resistance guidelines [14,15]. PCR amplification was performed under standard conditions: initial denaturation at 95°C for 5 min; 35 cycles of denaturation at 95°C for 30–60 s, locus-specific annealing (53.8–59°C, 30–60 s), and extension at 72°C for 30–60 s; followed by a final extension at 72°C for 10 min (Table 3). PCR products were verified by agarose gel electrophoresis. Amplicons were either sequenced immediately or stored at −20 °C until analysis. Sanger sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit and an ABI Prism 310 Genetic Analyzer. Sequence data were analyzed using the Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/BLAST) against the M. tuberculosis H37Rv reference genome (GenBank accession AL123456.3). SNV and other mutations identified from sequence alignments. Representative nucleotide sequences generated in this study have been provided as Supplementary File S1 for review purposes.

4.5. Statistical Analysis

MIC distributions were summarized as MIC₅₀, IQR, and MIC₉₀. Differences in MIC values between fully drug-susceptible and MDR-TB isolates were compared using the Mann–Whitney U test. Resistance proportions were expressed as percentages with 95% confidence intervals (CIs). A two-sided P < 0.05 was considered statistically significant.