ABSTRACT
Mycobacterium avium-intracellulare complex (MAC) infections are the most common nontuberculous mycobacterial infections, and ethambutol (EMB) is one of the main therapeutic agents used to treat MAC infections. However, the EMB resistance profile and its resistance mechanisms in M. avium remain poorly understood. This study determined the minimum inhibitory concentration (MIC) of 40 M. avium clinical strains, revealing that 97.5% (39/40) of the strains were intermediate (MIC = 4 µg/mL) or resistant (MIC ≥ 8 µg/mL) to EMB. One susceptible clinical isolate strain 245 (MIC = 2 µg/mL) was picked for EMB-resistant mutant isolation, and a total of 121 resistant mutants were isolated and subjected to whole-genome sequencing or Sanger sequencing. Integrated analysis revealed that 94.21% (114/121) of the mutants carried mutations in the ubiA gene, which encodes decaprenylphosphoryl-β-D-5-phosphoribose (DPPR) synthase—an enzyme involved in cell wall biosynthesis that has been associated with high-level EMB resistance in Mycobacterium tuberculosis. Complementation with the wild-type ubiA gene restored EMB susceptibility in EMB-resistant mutants and clinical strain 322 (EMB MIC = 64 µg/mL) with ubiA mutation, reducing the MICs from 32 to 4 μg/mL and from 64 to 8 μg/mL, respectively. This study indicates that the ubiA mutation is the major mechanism of EMB resistance in M. avium, which should facilitate the development of molecular tests for rapid detection of resistance in this organism.
IMPORTANCE
This study identified ubiA as a key gene associated with ethambutol (EMB) resistance in Mycobacterium avium, a finding which has not previously been reported. Furthermore, although ubiA has been linked to EMB resistance in Mycobacterium tuberculosis (MTB), mutations in this gene account for only a small proportion of EMB-resistant MTB isolates. In contrast, our study showed that ubiA played a major role in EMB resistance in M. avium. Our findings contribute to the development of molecular assays for rapid detection of EMB resistance in M. avium and highlight the distinct main resistance mechanisms across different bacterial species to the same antibiotic.
KEYWORDS: Mycobacterium avium, ethambutol, resistance mechanism, mutations, ubiA, minimum inhibitory concentration
INTRODUCTION
Mycobacterium avium complex (MAC) is a group of several slow-growing mycobacteria and is commonly found in the environment, such as water and soil (1, 2). MAC is primarily transmitted through aerosol inhalation and can cause infections, often in immunocompromised individuals (3). Patients who are aged, immunosuppressed, or have other respiratory diseases (such as cystic fibrosis, bronchiectasis, and COPD) are at higher risk of developing severe clinical symptoms following MAC infections (4–6). MAC comprises various subspecies, including M. avium, M. intracellulare, M. colombiense, M. timonense, etc., with M. avium being one of the most prevalent species globally (7). Current treatment guidelines for MAC infections recommend a multi-drug combination therapy with macrolides (clarithromycin/azithromycin) as the core drug, along with ethambutol (EMB), rifampicin (RIF), aminoglycosides or other drugs, and a three-drug regimen (macrolide, RIF, and EMB) is commonly recommended for the treatment of M. avium complex pulmonary disease (8).
EMB, as a key component in MAC treatment regimens, inhibits bacterial growth by interfering with arabinogalactan biosynthesis in the mycobacterial cell wall (9). Not only does its use significantly improve sputum conversion rates compared to RIF, streptomycin, or fluoroquinolones in the treatment of CLR-resistant MAC infections, but it also effectively prevents the development of macrolide resistance (10, 11). In patients with AIDS, EMB also had greater antibacterial activity than either RIF or clofazimine, supporting its unshakable position in the treatment of MAC infection (12). In terms of drug susceptibility, although a previous study classified MAC strains as susceptible (minimum inhibitory concentration [MIC] ≤ 2 µg/mL), intermediate (MIC = 4 µg/mL), or resistant (MIC ≥ 8 µg/mL) to EMB, many studies indicated that the MICs of M. avium strains of EMB are mainly 8 μg/mL, while the Clinical and Laboratory Standards Institute (CLSI) does not yet have EMB susceptibility or resistance breakpoints for M. avium (13–15). Furthermore, it has been suggested that when MAC isolates exhibit MICs ≥8 µg/mL for both EMB and RIF, the clinical treatment success rate will decrease significantly, indicating the negative correlation between successful treatment outcome with resistance to EMB and RIF (16). Therefore, elucidating the mechanism of EMB resistance in MAC remains clinically significant. To date, embB/embR aftA and ubiA genes have been proven to be associated with EMB resistance in Mycobacterium tuberculosis (MTB) (17–20), but the resistance mechanism in MAC is far less explored. Although previous studies have revealed that embA/B contribute to elevated EMB resistance in M. avium (21), clinical investigation indicated that embB mutations account for only about 4% of the EMB-resistant MAC clinical isolates (22), indicating other major EMB resistance mechanisms remain to be identified.
To better understand the mechanisms of EMB resistance in M. avium, here, we isolated EMB-resistant mutants and performed whole-genome sequencing and Sanger sequencing to investigate the potential EMB resistance mechanisms in M. avium.
MATERIALS AND METHODS
Strains and antibiotics
All the M. avium clinical strains were provided by The First Affiliated Hospital, Zhejiang University School of Medicine (Hangzhou, China). Clinical strains were identified as M. avium through whole-genome sequencing (Uni-medica, Shenzhen, China). Ethambutol (Aladdin, Shanghai, China) was dissolved in DMSO (Aladdin, Shanghai, China) and prepared as a 5.12 mg/mL stock solution and was stored at −20°C before use. Kanamycin sulfate (Aladdin, Shanghai, China) was dissolved in double-distilled water and prepared as a 100 mg/mL stock solution, and was stored at −20°C before use.
MIC determination and mutant screening
The MIC determination was performed using the microdilution method as previously described (23). The drug solution was diluted to 128 µg/mL in Middlebrook 7H9 broth (Difco, USA) supplemented with 5% OADC (oleic acid, albumin, glucose, and catalase) and 0.2% glycerol, and serial twofold dilutions (100 μL per well) were performed in 96-well plates (NEST, Wuxi, China). The bacterial suspension was diluted to 0.5 MacFarland Standard in Middlebrook 7H9 broth as mentioned above at first, then further diluted 100-fold, and a 100 μL aliquot of the suspension was added into each well with EMB, resulting in 11 drug concentrations ranging from 64 to 0.0625 μg/mL. The final bacterial count per well was approximately 5 × 10⁵ CFU/mL (ranging from 1 × 10⁵ to 1 × 106 CFU/mL). Positive control wells that contained only bacteria were set in the last column of the 96-well plates, and negative control wells that contained only the drug were set separately. The plates were incubated at 37°C for 2 weeks, and the results were read. The MIC test was performed in triplicate.
After MIC determination in 7H9 broth, the strain used for isolating EMB-resistant mutant strains was identified. Middlebrook 7H11 agar plates (Hopebio, Qingdao, China, containing 10% OADC and 0.5% glycerol) with EMB concentrations ranging from 1/4 to 4 times MIC were prepared, along with an antibiotic-free 7H11 agar plate as a control. The experimental approach for determining MIC on agar plates was described previously (24). A 20 μL of 1,000-fold diluted log-phase bacterial suspension was added to the 7H11 agar plates to confirm the MIC was consistent in both liquid and solid media. After 2 weeks of incubation at 37°C, the MIC on agar plates was determined.
For mutant screening, based on the MIC results from the 7H11 plates, 7H11 plates containing EMB at 4, 8, 16, and 32 times the MIC were prepared. A 100 μL aliquot of the stationary-phase bacterial suspension was then spread on the 7H11 EMB plates and incubated for 2 weeks to determine the optimal concentration to screen the EMB-resistant strains, which was used in the follow-up mutant screening. Single colonies of resistant mutants were picked and streaked onto 7H11 plates with a higher concentration of EMB, followed by incubation for another 2 weeks to make sure that the bacteria were resistant to EMB. The original sensitive strain was streaked onto the plate as a control.
Whole-genome sequencing
Genomic DNA was isolated from 10 mL of bacterial culture, and a number of resistant mutants and the original sensitive strain were picked. The cultures were centrifuged at 4,000 rpm for 10 min at 4°C, after which the supernatant was discarded. The pellets were washed twice with sterile water. The samples were sent for whole-genome sequencing using the Illumina platform (Novogene, Beijing, China). The genomic sequences of the resistant mutants were compared to those of the sensitive parent strain, and single-nucleotide polymorphisms (SNPs) were analyzed using software Snippy to identify putative mutations associated with EMB resistance in M. avium.
PCR and Sanger sequencing
The ubiA gene sequence was obtained from the whole-genome sequencing data of the susceptible strain by bacterial genome annotation software Bakta. Primers were designed as follows: ubiA_F: ATGACCGAGGAAACGCAGG; ubiA_R: CTAACCGAAGGCAACGGCG. Phanta Flash Master Mix (Dye Plus) (Vazyme, Nanjing, China) was used for PCR amplification, and the PCR conditions were as follows: denaturation at 98°C for 10 min, followed by 35 cycles of 98°C for 10 s, 60°C for 5 s, and 72°C for 5 s, with a final extension at 72°C for 1 min.
For all remaining resistant mutants, the original susceptible strain and all other clinical strains, a 918-base pair fragment containing the ubiA gene was subjected to Sanger sequencing (Tsingke, Beijing, China), and the sequences were aligned for comparison by SnapGene.
Gene complementation experiment
The target wild-type ubiA gene was amplified by PCR using the following primers: pmvubiA-F: caggaattcgatatcaagcttATGACCGAGGAAACGCAGG; pmvubiA-R: acgctagttaactacgtcgacCTAACCGAAGGCAACGGCG. The PCR product was purified by Fastpure Gel DNA Extraction Mini Kit (Vazyme). The pMV306hsp plasmid (25, 26) was digested with HindIII and SalI, followed by cloning of the ubiA PCR product into the linearized plasmid vector using ClonExpress Ultra One Step Cloning Kit (Vazyme). The recombinant plasmid was then transformed into Escherichia coli DH5α (Vazyme) by heat shock, and the transformant was incubated in a shaker at 37°C for 1 h, followed by plating onto LB agar plates (Hopebio, Qingdao, China) containing 100 µg/mL kanamycin and incubated at 37°C overnight. Single colonies were selected the next day, cultured in LB broth (Hopebio, Qingdao, China) supplemented with 100 µg/mL kanamycin, and verified by Sanger sequencing to confirm that the wild-type ubiA gene was successfully cloned into pMV306hsp. The wild-type ubiA recombinant plasmid was then extracted with the Fastpure Plasmid Mini Kit (Vazyme) and electroporated (2,500 V, 1,000 Ω, and 25 uF) into the EMB-resistant strains. The electroporated mutant strains were incubated in a 37°C shaker overnight and then plated onto 7H11 agar plates containing 100 µg/mL kanamycin and incubated at 37°C for 2 weeks. Single colonies that grew on the plates were then subjected to PCR sequencing to confirm that the wild-type ubiA gene was successfully introduced into the EMB-resistant strains. The complementation strains were tested for EMB susceptibility in 7H9 broth to validate the association between ubiA mutation and EMB resistance. Empty vector controls were included to exclude the potential effects of vector on MIC determinations.
RESULTS
Determination of EMB MICs for M. avium clinical strains
The EMB MIC values for 40 M. avium clinical strains were determined as described in the Materials and Methods. As summarized in Table 1, according to the previous study (13), only one clinical strain (strain 245) was susceptible to EMB (MIC ≤ 2 µg/mL), while all other clinical strains exhibited at least intermediate susceptibility (MIC ≥ 4 µg/mL) to EMB (97.5%, 39/40), and 82.5% of the clinical strains (33/40) were resistant (MIC ≥ 8 µg/mL) to EMB (13).
TABLE 1.
Ethambutol MIC distribution of M. avium clinical isolates
| Strain name | MIC (µg/mL) | Source | Total |
|---|---|---|---|
| 245 | 2 | Sputum | 1 |
| 118 | 4 | Sputum | 6 |
| 458 | 4 | Sputum | |
| 141 | 4 | Sputum | |
| 150 | 4 | Sputum | |
| 635 | 4 | Sputum | |
| 1055 | 4 | Urine | |
| 1006 | 8 | Sputum | 21 |
| 640 | 8 | Sputum | |
| 990 | 8 | Sputum | |
| 1237 | 8 | Sputum | |
| 1004 | 8 | Sputum | |
| 515 | 8 | Sputum | |
| 1224 | 8 | Sputum | |
| 142 | 8 | Sputum | |
| 715 | 8 | BALFa | |
| 1200 | 8 | Drainage fluid | |
| 938 | 8 | Sputum | |
| 1221 | 8 | Sputum | |
| 642 | 8 | Sputum | |
| 879 | 8 | Sputum | |
| 643 | 8 | Sputum | |
| 123 | 8 | Sputum | |
| 375 | 8 | Sputum | |
| 658 | 8 | Sputum | |
| 757 | 8 | Sputum | |
| 1015 | 8 | Sputum | |
| 1126 | 8 | Sputum | |
| 1083 | 16 | Drainage fluid | 10 |
| 1148 | 16 | Sputum | |
| 758 | 16 | Secretion | |
| 158 | 16 | Sputum | |
| 765 | 16 | Sputum | |
| 699 | 16 | Sputum | |
| 166 | 16 | Sputum | |
| 119 | 16 | Sputum | |
| 304 | 16 | Hydrothorax | |
| 374 | 16 | Sputum | |
| 860 | 32 | Drainage fluid | 1 |
| 322 | 64 | Sputum | 1 |
| Total | 40 |
BALF, bronchoalveolar lavage fluid.
Mutant isolation
From the above results, we selected the EMB-susceptible strain 245 (EMB MIC = 2 µg/mL) for isolating EMB-resistant mutants. Since the isolation of mutant strains was performed on 7H11 plates, the MIC of clinical strain 245 was reassessed on 7H11 agar plates containing EMB at concentrations ranging from 0.5 to 8 μg/mL, based on the previous MIC result obtained in 7H9 broth. The final EMB MIC of strain 245 on 7H11 agar plate was determined to be 4 μg/mL, which is twofold higher than its MIC in 7H9 liquid medium.
Based on the MIC results obtained from 7H11 agar plates, mutant isolation was conducted on 7H11 agar plates containing EMB at concentrations of 4× MIC and above, i.e., 16, 32, 64, and 128 μg/mL. After 2 weeks of incubation, it was determined that 16 μg/mL was the optimal concentration for mutant isolation, as 7H11 agar plates containing higher EMB concentrations failed to grow mutant colonies. The concentration of 16 μg/mL was subsequently used for large-scale screening of EMB-resistant mutants. All the colonies that grew on the plates containing 16 μg/mL were picked and streaked onto 7H11 agar plates containing EMB at 32 μg/mL together with the original susceptible strain. The mutants that grew both on plates containing 16 and 32 μg/mL EMB were regarded as authentic resistant mutants, and finally, a total of 121 mutants were successfully isolated.
Whole-genome sequencing and Sanger sequencing
To identify the potential mechanisms of EMB resistance in M. avium, whole-genome sequencing was performed initially on eight EMB-resistant mutants along with the parent susceptible strain as a control. The results showed that all the mutants harbored mutations in the ubiA gene, strongly implying an association between ubiA gene mutation and EMB resistance in M. avium. The remaining 113 resistant mutants were all subsequently subjected to ubiA Sanger sequencing. The results showed that most of the mutants harbored mutations in ubiA (93.81%, 106/113), which is consistent with and supports the initial whole-genome sequencing findings. Integrating the results from both whole-genome and Sanger sequencing, the mutations were found to be distributed throughout the ubiA gene. The majority of mutants carried single-site point mutations at different locations of the ubiA gene, accounting for 80.99% (98/121) of the observed mutations. However, 13.22% (16/121) of the mutants had multi-site mutations, insertions, and deletions in the ubiA gene (Table 2). Additionally, seven mutants (5.79%, 7/121) exhibited no mutations in the ubiA gene. To investigate other potential EMB resistance mechanisms in M. avium, we further performed whole-genome sequencing on these seven mutants lacking ubiA mutations. However, comparative genomic analysis revealed no mutations relative to the parent strain, implicating that their resistance mechanism remains unknown. The results of the ubiA mutations identified through both whole-genome sequencing and Sanger sequencing are summarized in Table 2.
TABLE 2.
The proportion of ubiA mutations at different sites in all EMB-resistant mutantsa
| Nucleotide mutation | Mutant type | Amino acid change | Number of the mutants | Proportion (%) |
|---|---|---|---|---|
| C113A | SNP | Ala38Glu | 2 | 1.65 |
| G121C | SNP | Ala41Pro | 6 | 4.96 |
| G124C | SNP | Ala42Pro | 3 | 2.48 |
| T128C | SNP | Leu43Pro | 7 | 5.79 |
| C495G | SNP | Ile165Met | 1 | 0.83 |
| G503A | SNP | Gly168Asp | 7 | 5.79 |
| T524C | SNP | Leu175Pro | 6 | 4.96 |
| A526G | SNP | Thr176Ala | 1 | 0.83 |
| C527T | SNP | Thr176Ile | 10 | 8.26 |
| A530T | SNP | Gln177Leu | 1 | 0.83 |
| T535C | SNP | Phe179Leu | 15 | 11.57 |
| T535A | SNP | Phe179Ile | 1 | 0.83 |
| T536C | SNP | Phe179Ser | 3 | 2.48 |
| T536G | SNP | Phe179Cys | 1 | 0.83 |
| C537G | SNP | Phe179Leu | 5 | 4.13 |
| C537A | SNP | Phe179Leu | 2 | 1.65 |
| T542C | SNP | Leu181Pro | 9 | 7.44 |
| T542A | SNP | Leu181Gln | 1 | 0.83 |
| T722C | SNP | Phe241Ser | 5 | 4.13 |
| T722G | SNP | Phe241Cys | 2 | 1.65 |
| G728A | SNP | Arg243His | 7 | 5.79 |
| G754T | SNP | Val252Leu | 2 | 1.65 |
| G754C | SNP | Val252Leu | 1 | 0.83 |
| T518A and C520G | MNP | Ile173Asn and Arg174Gly | 1 | 0.83 |
| Ins of AAC 527 | Insertion | FSC176 | 1 | 0.83 |
| Del of 100_102 CTG | Deletion | FSC34 | 1 | 0.83 |
| Del of 109_111 CTG | Deletion | FSC37 | 2 | 1.65 |
| Del of 115_123 CCGGTGGCG | Deletion | FSC39 | 1 | 0.83 |
| Del of 118_126 GTGGCGGCG | Deletion | FSC40 | 1 | 0.83 |
| Del of 124_126 GCG | Deletion | FSC42 | 6 | 4.96 |
| Del of 151_153 TAC | Deletion | FSC51 | 1 | 0.83 |
| Del of 757_759 GTG | Deletion | FSC253 | 2 | 1.65 |
| No mutations | 7 | 5.79 | ||
| Total mutants | 121 |
Ins, insertion; Del, deletion; FSC, frame shift codon; SNP, single nucleotide polymorphism; MNP, multiple nucleotide polymorphism.
Majority of M. avium clinical isolates resistant to EMB carry mutations in ubiA gene
To assess whether mutations in ubiA are associated with EMB resistance in M. avium clinical strains, we performed Sanger sequencing of the ubiA gene on the remaining 39 M. avium clinical strains (Table 1) and compared their sequences with that of strain 245, the EMB-susceptible strain. Sequencing results revealed nonsynonymous ubiA mutations in 32 of 39 (82.05%) clinical strains, while seven strains (six strains with EMB MIC ≥ 8 µg/mL, one strain with MIC = 4 µg/mL) (7 of 39 = 17.95%) did not have ubiA mutations. Among the 32 strains with ubiA mutations, 27 were resistant to EMB (MIC ≥ 8 µg/mL) and five exhibited intermediate susceptibility (MIC = 4 µg/mL), suggesting a correlation between ubiA variations and EMB susceptibilities in different M. avium clinical strains. The Sanger sequencing results for the ubiA gene in clinical strains are summarized in Table 3.
TABLE 3.
Mutations in the ubiA gene of M. avium clinical strains compared to the EMB susceptible strain 245
| Strain name | Number of strains | Nucleotide variations | Amino acid changes |
|---|---|---|---|
| 1126, 118, 141, 635, 1055, 1006, 640, 990, 1237, 1004, 515, 142, 715, 1200, 642, 643, 123, 375, 658, 1015, 1083, 1148, 758, 158, 765, 166, 304, 374, 322 | 29 | A169C | Lys57Gln |
| 1126, 118, 141, 635, 1055, 1006, 640, 990, 1237, 1004, 515, 142, 715, 1200, 642, 643, 123, 375, 658, 1015, 1083, 1148, 758, 158, 765, 166, 304, 374, 322 | 29 | G297A | Val99Val |
| 1126, 118, 141, 150, 635, 1055, 1006, 640, 990, 1237, 1004, 515, 142, 715, 1200, 642, 643, 123, 375, 658, 757, 1015, 1083, 1148, 758, 158, 765, 166, 119, 304, 374, 322 | 32 | A333G | Leu111Leu |
| 1126, 118, 141, 150, 635, 1055, 1006, 640, 990, 1237, 1004, 515, 142, 715, 1200, 642, 643, 123, 375, 658, 757, 1015, 1083, 1148, 758, 158, 765, 166, 304, 374, 322 | 31 | C525G | Leu175Leu |
| 1126, 118, 141, 150, 635, 1055, 1006, 640, 990, 1237, 1004, 515, 142, 715, 1200, 642, 643, 123, 375, 658, 757, 1015, 1083, 1148, 758, 158, 765, 166, 119, 304, 374, 322 | 32 | A766G | Ile256Val |
| 119 | 1 | G282C | Pro94Pro |
| 658, 1126, 158 | 3 | A451G | Met151Val |
| 119 | 1 | G711C | Gly237Gly |
| 119 | 1 | G816C | Gly272Gly |
| 757, 150 | 2 | C822T | Ala274Ala |
| 938, 879, 1224, 1221, 860, 699, 458 | 7 | No mutations |
Complementation of the EMB-resistant mutant with the wild-type ubiA restored EMB susceptibility
To determine whether the identified mutations in the ubiA gene contribute to EMB resistance, we subsequently introduced the wild-type ubiA gene into the randomly selected EMB-resistant mutants. Mutant strain 1 (mutation: G124C) and Mutant strain 19 (mutation: G754C) were used in the gene complementation experiment, with the empty vector being included as a control. The complemented mutants exhibited significantly reduced EMB MIC values (4 μg/mL) compared to mutants transformed with an empty vector control (32 μg/mL) (Table 4). To determine whether the role of ubiA in EMB resistance is consistent across different genetic backgrounds, we further electroporated the wild-type ubiA gene from strain 245 into strain 322, which exhibited the highest EMB MIC (64 μg/mL) among the clinical strains tested in our study (Table 1). Complementation with the wild-type ubiA gene also restored EMB susceptibility, reducing the EMB MIC in strain 322 from 64 to 8 μg/mL (Table 4). These results indicated that mutations in the ubiA gene are directly responsible for EMB resistance in diverse M. avium strains.
TABLE 4.
MICs of M. avium parent strain and ubiA mutant strains complemented with the wild-type ubiA gene
| Strain name | EMB MIC (µg/mL) |
|---|---|
| Parent strain 245 | 2 |
| Mutant strain 1 (ubiA mutation: G124C) | 32 |
| Mutant strain 1 (pMV306hsp vector alone) | 32 |
| Mutant strain 1 (pMV306hspWTubiA) | 4 |
| Mutant strain 19 (ubiA mutation: G754C) | 32 |
| Mutant strain 19 (pMV306hsp vector alone) | 32 |
| Mutant strain 19 (pMV306hspWTubiA) | 4 |
| Strain 322 (ubiA mutations: A169C, A766G) | 64 |
| Strain 322 (pMV306hsp vector alone) | 64 |
| Strain 322 (pMV306hspWTubiA) | 8 |
DISCUSSION
Although the use of EMB in the treatment of MAC infections is now widely accepted, the mechanisms underlying EMB resistance in this pathogen remain poorly characterized (8). In this study, we screened 121 EMB-resistant mutants of M. avium and performed whole-genome sequencing and targeted ubiA Sanger sequencing to investigate the genetic basis of resistance of M. avium to EMB. Integrated analysis of both whole-genome sequencing and ubiA sequencing data revealed that 94.21% (114/121) of the EMB-resistant strains harbored mutations in the ubiA gene, indicating that mutations in ubiA are the major mechanism of EMB resistance in M. avium. Furthermore, genetic complementation experiments confirmed that ubiA is the causative of EMB resistance in M. avium.
ubiA, which encodes decaprenylphosphoryl-β-D-5-phosphoribose (DPPR) synthase and corresponds to Rv3806c in M. tuberculosis, is involved in the synthesis of decaprenylphosphoryl-β-D-arabinose (DPA). DPA serves as the donor substrate for arabinosyltransferases, including EmbB and EmbC, which are involved in catalyzing the transfer of arabinose from DPA to form the arabinan components of the mycobacterial cell wall (19, 27). Previous studies indicate that in strains with a wild-type ubiA genotype, EMB inhibits EmbCAB activity, thereby disrupting cell wall integrity and resulting in bacterial death (28, 29). However, when ubiA mutates, ubiA mutations will increase DPA levels, and the elevated DPA will functionally counteract EMB inhibition by providing an excess of the substrate for the EmbCAB, reducing EMB inhibition and conferring high-level resistance in MTB (20). Given the close phylogenetic relationship between M. avium and MTB, it is plausible that ubiA mutations modulate EMB susceptibility through similar mechanisms in both species. Nevertheless, a striking aspect of our findings is that M. avium and MTB acquire EMB resistance through mutations in different genes, despite using the same arabinan biosynthetic pathway. In MTB, resistance is overwhelmingly mediated by mutations in embB, which encodes the arabinosyltransferase directly bound and inhibited by EMB (30, 31). In MTB, ubiA mutations actually account for a relatively small proportion of EMB-resistant strains, and embB mutations are far more common (32). Even in high-level EMB-resistant MTB strains only, the proportion of ubiA mutation occurrence fluctuates with changes in geographical location but mostly does not exceed 10%, especially in Asian countries (33–36). In contrast, our results demonstrate that M. avium acquires resistance primarily through mutations in ubiA, a gene encoding DPPR, which produces the precursor (DPPR/DPA) required for arabinan synthesis, as mentioned above. Unlike MTB, ubiA mutations were identified in more than 90% of EMB-resistant M. avium mutants selected in this study, and 82.05% (32/39) of the M. avium clinical strains, which were all EMB-intermediate/resistant, also harbored ubiA mutations, further demonstrating a fundamental clinical difference in EMB resistance between the two species. This species difference likely reflects distinct pathway bottlenecks and metabolic flux control points in the two organisms. In MTB, direct modification of the EMB target (EmbB) is the most effective and lowest-cost route to resistance. However, in M. avium, mutations that increase precursor (DPA) production appear to be strongly favored: elevated DPA levels can competitively displace EMB from EmbB/EmbC, reducing drug efficacy even when the target remains wild type. Thus, although the two organisms share the same cell-wall arabinan pathway, the adaptive landscape of EMB resistance differs, with MTB selecting for target modification, and M. avium selecting for altered precursor supply. Future comparative studies are needed to elucidate the different antibiotic resistance mechanisms across bacterial species, which could inform the development of species-specific molecular diagnostics.
Apart from the difference in the primary resistance mechanism to EMB between the MTB and M. avium, it is noteworthy that seven EMB-resistant mutants in this study lacked mutations in the ubiA gene. Furthermore, Sanger sequencing of the ubiA gene in all 39 clinical strains revealed that seven clinical strains, six of which exhibited EMB MICs ≥8 µg/mL and one exhibited 4 μg/mL (Tables 1 and 3), had no ubiA mutations compared with the susceptible control strain 245 (MIC = 2 µg/mL). These findings strongly suggest the existence of alternative, as yet unidentified resistance mechanisms in M. avium, which warrant further investigations.
It is also worth noting that in our MIC assays, nearly all clinical strains exhibited resistance (MIC ≥ 8 µg/mL) or intermediate susceptibility (MIC = 4 µg/mL) to EMB, based on breakpoints established in an earlier study (13). Our MIC test results are consistent with the previous literature, enhancing the reliability of our results (37). This finding mentioned above stands in sharp contrast to the resistance profile observed in M. tuberculosis, where only 5.7% of newly diagnosed patients and 17.2% of re-treated patients show EMB resistance under the CLSI guidelines, which define susceptibility as MIC ≤2 µg/mL and resistance as MIC ≥8 µg/mL (38–40). The discrepancy of the EMB resistance rate between MTB and M. avium suggests that wild-type M. avium may commonly possess intrinsic resistance mechanisms against EMB, or the EMB’s susceptibility/resistance breakpoint for M. avium should be modified.
Another interesting point, although not associated with drug resistance, is that mutations in the ubiA gene do not confer antibiotic resistance to M. abscessus (including EMB) but can trigger a greater pro-inflammatory response (41). Concurrently, these mutations help the bacterium evade immune cell uptake—an effect that is dependent on both the bacterial morphotype and the type of immune cell—and promote biofilm formation, which collectively facilitates the establishment of chronic lung infection (41). Whether ubiA mutations play a similar role in establishing a chronic infection by M. avium remains to be determined, and it warrants future studies to comprehensively understand the potential diverse effects of ubiA on M. avium infection.
This study has several limitations. Firstly, as the ubiA gene is primarily associated with high-level EMB resistance, and all mutants in this study were selected on 7H11 agar plates containing 32 µg/mL EMB, our approach may have limited the detection of mutations conferring low-level EMB resistance. Secondly, most of the work in our study was conducted using a single clinical strain, and future studies should include a broader collection of EMB-resistant clinical isolates to further examine the correlation between ubiA mutations and EMB resistance in M. avium. Thirdly, we did not further investigate the resistance mechanisms of EMB-resistant clinical strains without ubiA gene mutations due to their different genetic backgrounds, making it difficult to identify the resistance mutations.
In conclusion, our study revealed that, unlike MTB, where embB is the main resistance determinant, ubiA mutations are the major mechanism of EMB resistance in M. avium, which has not been reported before. The finding that ubiA mutations are the primary mechanisms of EMB resistance in M. avium has implications for the rapid detection of EMB-resistant M. avium strains in the future. Further studies are still needed to investigate the alternative mechanisms of EMB resistance in M. avium.
ACKNOWLEDGMENTS
This study was supported by the National Infectious Disease Medical Center startup fund (Y.Z.) (B2022011-1).
Contributor Information
Ying Zhang, Email: yzhang207@gdmu.edu.cn.
Selvakumar Subbian, Rutgers New Jersey Medical School, Newark, New Jersey, USA.
DATA AVAILABILITY
The whole-genome sequence data for the original strain 245 and its 8 mutant strains with ubiA mutations that support the findings of this study are submitted to the NCBI database with BioProject ID PRJNA1417368.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The whole-genome sequence data for the original strain 245 and its 8 mutant strains with ubiA mutations that support the findings of this study are submitted to the NCBI database with BioProject ID PRJNA1417368.